Novel feline cytokine protein

ABSTRACT

A novel feline cytokine protein having the activity to enhance the cytotoxic activity of feline cytotoxic T lymphocytes, a DNA sequence coding for said protein, a recombinant DNA for expressing said protein, an expression vector comprising said recombinant DNA, a transformant which is transformed with said expression vector, a process for preparing said protein by culturing said transformant, and an antibody against said protein are provided. The novel feline cytokine protein of the present invention is a heterologous dimer comprising FLAF p35 and FLAF p40 and can be used for treating feline infectious diseases such as feline herpes virus type 1 (FHV-1) or feline calicivirus (FCV).

TECHNICAL FIELD

[0001] The present invention relates to a novel polypeptide tide, anovel protein comprising a homologous dimer or a heterologous dimer ofsaid polypeptide, a novel DNA coding for said peptide, a recombinant DNAmolecule comprising said DNA, a transformant which is transformed withsaid recombinant DNA molecule, an antibody against said novelpolypeptide or said novel protein, a process for preparing said novelpolypeptide or said novel protein, and an agent for treating felineviral diseases comprising said novel protein or said novel antibody.More particularly, the present invention relates to a feline cytokineprotein comprising two distinct novel polypeptides having an activity todamage virus-infected cells by activating feline cytotoxic T lymphocytesand a gene coding for said cytokine protein as well as a process forpreparing said feline cytokine protein.

BACKGROUND ART

[0002] A cat is such an animal that has been loved by humans as a petfrom ancient times and, in modern times, called as Companion species, isbecoming a member of a human society. On the other hand, a cat hashitherto greatly contributed to humans as an experimental animal invarious fields such as medicine, pharmaceutics, animal husbandryveterinary and psychology, and in recent years, the contribution of acat has further increased to be used in an effectiveness assay or safetytest for drugs. With increasing social significance of a cat, there is ahigh interest in feline diseases, especially feline infectious diseases,and thus more efficacious method for treating these diseases is desired.

[0003] Many feline viral diseases, attack of which often leads toserious conditions, are known. For example, an upper tracheal diseasecaused by feline herpes virus type 1 (FHV-1) or feline calicivirus (FCV)is acute and highly lethal. In addition to this, diseases caused byfeline immunodeficiency virus, feline infectious peritonitis virus,feline parvovirus, etc. are also highly lethal and have been greatconcern. Although some prophylactic vaccines have been developed forthese viral diseases, many of these vaccines are not fully efficaciousdue to diversity of viral serotype. Furthermore, once a cat is infectedwith virus and after the onset of viral diseases, vaccines are notsubstantially efficacious any more, and hence, protection from secondarybacterial infections with antibiotics, sulfonamides etc. or symptomatictreatment with vitamins or nutrients have primarily been carried out.That is, presently no efficacious medicaments are available for treatingthe viral diseases.

[0004] Host immunity to microorganisms including viruses consists of ahumoral immunity by an antibody and a cellular immunity by a lymphocyte.A cellular immunity reaches at its maximum level seven to ten days afterinfection and thereafter declines rapidly. On the other hand, antibodyproduction starts to increase a week after infection, reaches at itsmaximum level around three to four weeks after infection and thereafterdeclines slowly. Since a neutralizing antibody is effective at the earlystage of viral infection as well as passive immunity, the crux ofdeveloping a vaccine for prophylaxis of diseases is to produce aneutralizing antibody in any way. Once infection has been established,however, a neutralizing antibody does not usually function effectivelywith the lapse of time after infection. Moreover, a neutralizingantibody is not so effective in case of a persistent infection (e.g.herpes virus) wherein a virus remains within cells and infects throughcells to cells or in case that a virus is likely to mutate even afterinfection to exhibit resistance against a neutralizing antibody (e.g.immunodeficiency virus). In such cases, it is generally believed thatonly a cellular immunity is effective which is mediated by cytotoxic Tlymphocytes (CTL) which find out and eliminate those cells infected withvirus. In general, a cytotoxic activity declines as symptoms becameserious with the lapse of time after infection, resulting in moreserious symptoms. In this context, if cytotoxic T lymphocytes could beactivated, then the progress of disease can be retarded and rapidrecovery will be expected (A. Capron et al., Current Topics inMicrobiology and Immunology 189 Springer-Verlag, 1994).

[0005] There is a human therapy by activation of cytotoxic T lymphocyteswherein cytotoxic T lymphocytes are recovered from peripheral blood and,after nonspecific activation in vitro, reintroduced into the livingbody. In case of cats, however, this therapy is not applicable sincesufficient amount of cytotoxic T lymphocytes cannot be recovered fromperipheral blood due to a small body size of cats. That is, at present,there is not found any therapy for effective treatment of feline viralinfectious diseases through activation of cytotoxic T lymphocytes.Although significance of a cellular immunity for treating viralinfectious diseases is suggested, there has not hitherto been reported acellular immunity-based medicament for feline viral infectious diseases,especially a medicament for activating cytotoxic T lymphocytes.

[0006] A cytokine is possibly involved in activation of cytotoxic Tlymphocytes. Feline cytokines reported hitherto include, for example,erythropoietin (D. Wen et al., Blood Vol. 82, 1507-1516, 1993), αinterferon (J. T. Yamamoto et al., Vet. Immunol. Immunopathol. Vol. 11,1-19, 1986), and the like. However, there has hitherto been no reportthat any of these cytokines activated cytotoxic T lymphocytes. On theother hand, human or mouse interleukin 12 (hereinafter referred to as“IL12”) is known which has an ability to activate T lymphocytes or toinduce γ interferon and activates cytotoxic T lymphocytes. Human andmouse IL12s cloned hitherto are known to exhibit the activity with aheterologous dimer consisting of an alpha chain (p35) and a beta chain(p40) (Annu. Rev. Immunol. Vol. 13, 251-276, 1995). It is also reportedthat human and mouse IL12s show species specificity (S. F. Wolf et al.,J. Immunol. Vol. 136, 3074-3081, 1991). As to feline IL12, a partialamino acid sequence of p35 has been reported (K. Bush et al., MolecularImmunology, Vol. 31, 1373-1374, 1994) with no disclosure of a biologicalactivity of IL12 while an amino acid sequence of p40 has not yet beenreported. Accordingly, at present, there is no substantial report as tofeline cytokines which activate cytotoxic T lymphocytes.

DISCLOSURE OF THE INVENTION

[0007] Under the circumstances, the present inventors have thoroughlystudied in order to find out feline cytokines which activate cytotoxic Tlymphocytes, and as a result, have found a proteinaceous agent (novelfeline cytokine) which enhances a cytotoxic activity of felinelymphocytes (especially cytotoxic T lymphocytes: CTL) in a culturesupernatant of activated feline splenocytes. Furthermore, the presentinventors have isolated and purified the novel feline cytokine proteinfrom the recovered culture supernatant, elucidated their properties,expressed them in an animal cell with a gene engineering technique, andfound that the resulting novel feline cytokine protein had an activityto enhance the cytotoxic activity of feline cytotoxic T lymphocytes,thereby completing the present invention.

[0008] That is, an object of the present invention is to provide a novelfeline cytokine protein which enhances a cytotoxic activity of felinecytotoxic T lymphocytes as well as a polypeptide comprising a partialamino acid sequence of said novel feline cytokine protein.

[0009] Another object of the present invention is to provide a genecoding for a novel feline cytokine protein which enhances a cytotoxicactivity of feline cytotoxic T lymphocytes or a polypeptide comprising apartial amino acid sequence of said novel feline cytokine protein aswell as a recombinant DNA molecule for expressing said gene.

[0010] Further object of the present invention is to provide a processfor preparing a novel feline cytokine protein which enhances a cytotoxicactivity of feline cytotoxic T lymphocytes and a polypeptide comprisinga partial amino acid sequence of said novel feline cytokine protein frommicroorganisms or animal cells transformed with said recombinant DNAmolecule.

[0011] Still further object of the present invention is to provide amonoclonal antibody and a polyclonal antibody produced by using as animmunogen the thus obtained novel feline cytokine protein or thepolypeptide comprising a partial amino acid sequence of said novelfeline cytokine protein.

[0012] Still another object of the present invention is to provide anagent for treating feline viral infectious diseases comprising as anactive ingredient the novel feline cytokine protein which enhances acytotoxic activity of feline cytotoxic T lymphocytes.

[0013] The novel feline cytokine proteins as found out by the presentinventors are a protein with a molecular weight of about 40,000 havingthe amino acid sequence as shown in Sequence Listing, SEQ ID NO: 1 orSEQ ID NO: 2 (hereinafter also referred to as “FLAF p40”), and a proteinwith a molecular weight of about 35,000 having the amino acid sequenceas shown in SEQ ID NO: 3 or SEQ ID NO: 4 (hereinafter also referred toas “FLAF p35”). The present invention also encompasses a peptidecomprising a partial amino acid sequence of these proteins.

[0014] The present invention also encompasses a polyclonal antibody anda monoclonal antibody prepared by using as an immunogen the novel felinecytokine protein or the peptide having a partial amino acid sequence ofsaid protein. Furthermore, the present invention encompasses an antibodyobtained by introducing an expression vector wherein a gene coding forthe polypeptide or a part of said gene is incorporated into an animalbody in a conventional manner for expression within said animal body.

[0015] The present invention also encompasses gene fragments which codefor the novel feline cytokine protein and have the nucleotide sequenceas shown in SEQ ID NO: 5, 6 or 7 for FLAF p40 or the nucleotide sequenceas shown in SEQ ID NO: 8 or 9 for FLAF p35, a gene fragment coding for apeptide comprising a partial amino acid sequence of said protein, aswell as a recombinant DNA molecule comprising these gene fragments.

[0016] In addition, the present invention encompasses a transformant (E.coli, yeast, an insect cell, an animal cell, a plant cell) transformedwith an expression vector such as a plasmid comprising the recombinantDNA molecule. The present invention also encompasses a process forpreparing a desired novel feline cytokine protein or a peptidecomprising a partial amino acid sequence of said protein by using saidtransformant.

BRIEF EXPLANATION OF THE DRAWINGS

[0017]FIG. 1 shows the CTL enhancing activity observed in culturesupernatant of feline splenocytes with culture supernatant of felinekidney cells being used as a negative control.

[0018]FIG. 2 shows the γ interferon inducing activity observed inculture supernatant of feline splenocytes with culture supernatant offeline kidney cells being used as a negative control.

[0019]FIG. 3 illustrates a pattern obtained by fractionation ofconcentrated culture supernatant of feline splenocytes by an anionexchange chromatography (MonoQ), which depicts that both elutionpatterns of the γ interferon inducing activity and the ⁵¹Cr releasingactivity are over-lapping.

[0020]FIG. 4 illustrates a pattern obtained by fractionation ofconcentrated culture supernatant of feline splenocytes by an anionexchange chromatography (MonoQ), which depicts that those fractions withthe γ interferon inducing activity are eluted with NaCl at aconcentration ranging from 200 mM to 300 mM.

[0021]FIG. 5 illustrates a pattern obtained by fractionation of thefractions obtained in FIG. 4 by a cation exchange chromatography (SPToyopearl), which depicts that those fractions with the γ interferoninducing activity are eluted with NaCl at a concentration ranging from500 mM to 600 mM.

[0022]FIG. 6 illustrates a pattern obtained by fractionation of thefractions obtained in FIG. 5 by a heparin column chromatography (HiTrapHeparin), which depicts that those fractions with the γ interferoninducing activity are eluted with NaCl at a concentration ranging from600 mM to 800 mM.

[0023]FIG. 7 illustrates a pattern obtained by fractionation of thefractions obtained in FIG. 6 by a gel filtration chromatography(HiLoad), which depicts that those fractions with the γ interferoninducing activity are eluted as fractions having a molecular weightranging from 60,000 to 80,000.

[0024]FIG. 8 is a photograph showing the results of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the fractionswith the γ interferon inducing activity obtained in FIG. 7 aftertreatment with 2-mercaptoethanol, which depicts both p40 and p35associated with the activity.

[0025]FIG. 9 shows a nucleotide sequence overlapping between 2 clones(clones 12 and 25) obtained after screening of cDNA library as well as acorresponding amino acid sequence.

[0026]FIG. 10 illustrates an expression vector for animal cell,pCAGn-mcs polyA, which contains human cytomegalo-virus enhancer, chickenβ actin promoter and rabbit β globin splice acceptor as an expressioncontrol region.

[0027]FIG. 11 is a photograph which proves the presence of a desiredexpression product when culture supernatant of transformed COS cells istreated with 2-mercaptoethanol, subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a thinmembrane wherein culture supernatant of COS cells transfected withpCAGn-mcs polyA is used as a negative control.

[0028]FIG. 12 shows the γ interferon inducing activity observed inculture supernatant of COS cells incorporated with expression vectors ofFLAF p40, feline IL12 p35 or FLAF p35, each alone or in combinationthereof, wherein culture supernatant of COS cells incorporated with anexpression vector comprising no exogenous gene is used as a negativecontrol.

[0029]FIG. 13 shows a nucleotide sequence of FLAF p35 (clone 20)obtained by screening of cDNA library as well as a corresponding aminoacid sequence, which depicts a region of the amino acid sequence that isdistinct from the reported feline IL12 p35.

[0030]FIG. 14 shows that culture supernatant of COS cells incorporatedwith both FLAF p40 and FLAF p35 expression vectors exhibits the CTLenhancing activity equivalent to that of culture supernatant of felinesplenocytes.

[0031]FIG. 15 shows that SFT34 cells incorporated with both FLAF p40 andFLAF p35 expression vectors stably produce FLAF at a clone level.

[0032]FIG. 16 is a photograph showing the results of testing forreactivity with serum from rabbit administered with CAGfp40 whereinpurified FLAF is subjected to sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol and thentransferred to a thin membrane where reactivity with the serum isinvestigated.

[0033]FIG. 17 shows an efficacy of FLAF for treating cats infected withfeline herpes virus wherein the axis of abscissas depicts days aftercompulsory infection, the axis of ordinates depicts a total of clinicalscore and “n” means a number of animals per group.

[0034]FIG. 18 is a photograph showing the results of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified FLAFin the absence of 2-mercaptoethanol wherein the gel is dyed withCoomassie Brilliant Blue.

[0035]FIG. 19 shows the γ interferon inducing activity of p75, p80 andpurified FLAF extracted from the gel wherein PBS is used as a negativecontrol.

BEST MODE FOR PRACTICING THE INVENTION

[0036] The present invention is explained in more detail hereinbelow.

[0037] The novel feline cytokine protein of the present invention can bepurified by culturing feline lymphocytes in the presence of a lymphocytemitogen and using as an index of purification the activity of cytotoxicT lymphocytes from the recovered culture supernatant.

[0038] For feline lymphocytes, splenocytes are preferably used butperipheral blood monocytes (PBMC) or some blood cancer cells may also beused. These cells may be cultured in a conventional procedure for cellculture, for example, as described in Culture of Animal Cells 2nd Ed.,Alan R. Liss, Inc., 1987. A lymphocyte mitogen includesphytohemagglutinin, concanavalin A, lipopolysaccharide, poke-weedmitogen with poke-weed mitogen (PWM) being preferred.

[0039] For measuring the activity of cytotoxic T lymphocytes as an indexof purification, several methods are known and available. The mostdirect method is a ⁵¹Cr release assay wherein the activity of cytotoxicT lymphocytes to destroy ⁵¹Cr labelled target cells is measured.Indirectly, γ interferon released when cytotoxic T lymphocytes areactivated can be measured. In these assay systems, it is desirable touse feline peripheral blood lymphocytes (PBL) as target cells. However,the use of feline PBL is not suitable for (1) CTL assay requiringpersistently infected cells since most of the viruses known at presentcause cytolysis after infection, and for (2) an assay system at eachpurification step which requires measurement of many samples sincelymphocytes obtainable from an individual cat is not abundant in numberwhile much more fresh peripheral blood monocytes (PBMC) are required foreach assay.

[0040] Under the circumstances, the present inventors have found thatthe CTL assay (⁵¹Cr release assay) system with human PBMC which has beenestablished for influenza virus (Gorse et al., J. Clinical Microbiology,Vol. 28, 2539-2550 (1990)) can be utilized for measuring the activity ofthe feline cytokine protein to activate lymphocytes. A target virus tobe used in this assay system is not limited to influenza virus but humanherpes simplex virus (HSV) may also be used. However, usable virus isnot limited to HSV but any virus which persistently infects duringmeasurement of the CTL activity may be used.

[0041] The novel feline cytokine protein of the present invention may beisolated and purified using the ability to activate lymphocytes as anindex by the usual methods employed in protein chemistry such as asalting out, an ultrafiltration, an isoelectric precipitation, anelectrophoresis, an ion exchange chromatography, a hydrophobicchromatography, a gel filtration chromatography, a reverse phasechromatography, an affinity chromatography, and the like. Preferably,the novel feline cytokine protein is purified by successively conductingan anion exchange chromatography, a cation exchange chromatography, aheparin column chromatography, and a gel filtration chromatography underthe conditions described in Example 2. According to these procedures,the novel feline cytokine protein can be purified up to 13% of anactivity yield and a higher specific activity by 2350 folds with anindex of the γ interferon inducing activity.

[0042] The thus obtained purified fractions are then subjected to sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in thepresence of 2-mercaptoethanol ethanol to give unique bands associatedwith the γ interferon inducing activity, i.e. a main band of a molecularweight about 40,000 (FLAF p40) and a minor band of about 35,000 (FLAFp35) (cf. FIG. 8). An amino acid sequence of these unique bands can bedetermined by a usual method such as Edman degradation (P. Edman, ActaChem. Scand., 4, 283 (1950)). As a result, FLAF p40 proved to be a novelfeline protein. On the other hand, FLAF p35 was confirmed to be apeptide comprising the feline IL12 p35, to which C terminal is attached19 amino acid residues:

[0043]Ala-Phe-Arg-Ile-Arg-Ala-Val-Thr-Ile-Asn-Arg-Met-Met-Ser-Tyr-Leu-Asn-Ser-Ser

[0044] as shown in SEQ ID NO: 10 as compared with the amino acidsequence of feline IL12 p35 disclosed in Bush et al. supra, It was alsofound that the signal sequence included substitution of an amino acid attwo sites (⁻²¹Arg, ⁻²⁰Gly; the numbering of an amino acid residue was inaccord with SEQ ID NO: 3) and insertion of 3 amino acid residues:

[0045] Asn-His-Leu

[0046] at the C terminal of ⁻¹⁰Leu (FIG. 13).

[0047] FLAF p40 and FLAF p35 genes may be cloned in a usual manner bypreparing mRNA and cloning cDNA as taught by Maniatis et al. (Molecularcloning, A Laboratory Manual 2nd Ed., 12.30, Cold Spring HarberLaboratory Press, N.Y., 1989). Briefly, a whole RNA is prepared fromfeline lymphocytes cultured in the presence of a lymphocyte mitogen, acDNA library is prepared therefrom, and FLAF p40 and FLAF p35 genes arescreened from the library by using synthetic oligonucleotides preparedbased on a partial amino acid sequence of FLAF p40 and FLAF p35 as aprobe. A lymphocyte mitogen includes phytohemagglutinin, concanavalin A,lipopolysaccharide, poke-weed mitogen with poke-weed mitogen (PWM) beingpreferred. Feline lymphocytes include splenocytes, peripheral bloodmonocytes (PBMC), some blood cancer cells, and the like, with felinesplenocytes being preferred. As a preferable probe, oligonucleotidesdesigned on the basis of the N terminal 20 amino acid sequence of FLAFp40 and the N terminal 10 amino acid sequence of FLAF p35, respectively.Alternatively, each of FLAF p40 and FLAF p35 proteins is subjected tolimited proteolysis, the resulting peptides are isolated by a reversephase chromatography, and an N terminal amino acid sequence isdetermined and used for designing oligonucleotides. As to FLAF p35,since the N terminal sequence is identical to that of feline IL12 p35(K. Bush et al., Molecular Immunology, Vol. 31, 1373-1374 (1994)), it isalso possible to utilize the sequence to design an oligonucleotide or aPCR primer and the PCR products may be used.

[0048] A nucleotide sequence of the thus cloned FLAF p40 and FLAP p35cDNAs may be determined with a DNA sequencer (Applied Biosystems, Model1373A). Novelty of these cDNAs may be confirmed by investigatinghomology between the whole nucleotide sequence of these cDNAs and theknown sequences in data base (for example, GeneBank, SWISS-PROT, etc.).

[0049] FLAF p40, FLAF p35, or a portion of each of these peptides, aheterologous dimer of FLAF p40 and FLAF p35 (hereinafter often referredto as “FLAF”), or a homologous dimer of FLAF p40 may be produced bypreparing a recombinant DNA molecule wherein a full length of FLAB p40cDNA, FLAF p35 cDNA, or cDNA fragment comprising a portion of the cDNAsis incorporated into a suitable expression vector, transforming asuitable microorganism or an animal cell with the recombinant DNAmolecule, and culturing the resulting transformant. A peptidesynthesizer may also be used for producing a portion of FLAF p40 or FLAFp35.

[0050] A suitable signal sequence for secretion in a microorganism or ananimal cell may be linked upstream of the DNA coding for the protein ofthe present invention so that said protein is secreted into a culturemedium. Such a modified DNA for secretion is advantageous in that theprotein secreted into the culture medium can easily be recovered. Asignal sequence includes pel B signal (S. P. Lei, et al., J.Bacteriology, Vol. 169, 4379-4383, 1987) for E. coli, β factor signal(A. J. Brake, Yeast Genetic Engineering, p269, Butterworth, 1989) foryeast, a signal of immunoglobulin, e.g. SG-1 antibody (H. Maeda et al.,Hum. Antibod. Hybridomas, Vol. 2, 124-134, 1991) and C25 antibody(patent, International Publication No. WO94/20632) for an animal cell,and the like. However, not limited to these, any signal sequence may beemployed insofar as it functions as a signal sequence.

[0051] An expression vector includes a plasmid, a viral vector, and thelike. A promoter to be contained in the expression vector may be anypromoter which is selected depending on a microorganism or an animalcell used as a host so that an active FLAF is ultimately obtained,including SV40 early promoter, SV40 late promoter, β actin promoter,etc. A marker gene may be used including an ampicillin resistance geneor a tetracycline resistance gene for E. coli or Leu2 gene for yeast incase of an expression vector for a microorganism. In case of anexpression vector for an animal cell, aminoglycoside3′-phosphotranferase (neo) gene, dihydrofolate reductase (dhfr) gene,glutamine synthetase (GS) gene and the like may be used. FIG. 10illustrates pCAGn-mcs polyA by way of example of such an expressionvector. A typical substance added for selection includes G-418,methotrexate, methionine sulphoximine, and the like.

[0052] In case of an expression vector for an animal cell, a host cellincludes various cells such as Chinese hamster ovary (CHO) cell, mousemyeloma cell, and COS cell. With a combination of the thus constructedFLAF p35 cDNA and FLAF p40 cDNA expression vectors, FLAF may transientlybe expressed in, for example, COS7 cell (derived from African greenmonkey) or stably expressed in SFT34 cell (patent, Internationalpublication No. WO94/12658) or CHO cell as a host.

[0053] A host cell is transformed by a known method including, forexample, a calcium phosphate method, a DEAE dextran method, aprecipitation method using lipofectin etc., a method of fusingprotoplasts with polyethylene glycol, a electroporation method, and thelike. A suitable method for transformation may be selected depending ona host cell used.

[0054] The novel feline cytokine protein of the present invention isprepared in the manner described below. The cells stably expressing FLAFare cultured under conditions usually used for lymphocytes, for example,in R?MI medium containing 1 mg/ml G418 and 10% FCS in case thatG418-resistant gene (Neo gene) is used as a selection marker, to giveG418-resistant cells, and FLAF-producing transformants are screened byan assay using the γ interferon inducing activity as an index. Ifnecessary, cloning is made by a limiting dilution technique to isolatesaid FLAF-producing clones. The thus obtained FLAF-producingtransformants are cultured in a large scale, and FLAF is purified fromthe recovered culture supernatant in a similar manner to the aboveprocedures by using as an index an immunochemical activity such as theactivity of cytotoxic T lymphocytes or an antibody against FLAF so thatthe novel feline cytokine protein is produced.

[0055] The novel feline cytokine protein thus obtained has the enhancedactivity to enhance the cytotoxic activity of feline cytotoxic Tlymphocytes. The novel feline cytokine protein is used as an agent fortreating feline viral infectious diseases or as an anti-cancer agent,which alone or in combination with a suitable pharmaceuticallyadministrable carrier, diluent or stabling agent, is conventionallyformulated into dosage forms such as injections, oral drugs,suppositories, or eye drops.

[0056] The novel feline cytokine protein of the present invention can beused for treatment of feline viral diseases including, for example, anupper tracheal disease caused by feline herpes virus type 1 (FHV-1) orfeline calicivirus (FCV), viral infectious diseases with felineimmunodeficiency virus, feline infectious peritonitis virus, felineparvovirus, feline panleukopenia virus, or feline leukemia virus. Thenovel feline cytokine protein of the present invention can also be usedas an anti-tumor agent or a parasiticidal agent due to its activity toactivate cytotoxic T lymphocytes.

[0057] The novel feline cytokine protein or a polypeptide comprising apartial amino acid sequence of said protein may be used as an immunogenfor preparing a polyclonal antibody or a monoclonal antibody by theconventional method now established. An expression vector wherein a genecoding for the polypeptide or a portion of said gene is incorporated maybe introduced into the animal body where said gene is expressed so thatan antibody against said polypeptide or partial peptide of saidpolypeptide is produced. The thus produced antibody, which binds to thenovel feline cytokine protein or a polypeptide comprising a partialamino acid sequence of said protein, or said protein or said polypeptidemay be used in a system for detecting an antigen such as Western blot orELISA, and thus constitutes a diagnostic agent. The antibody may bebound to a suitable carrier to provide an affinity chromatography whichcan be used to purify the antigenic protein.

[0058] The present invention provides a heterologous dimer proteincomprising FLAF p35 and FLAF p40 which exhibits the γ interferoninducing activity and the CTL enhancing activity as well as cDNAs codingfor each of these proteins.

[0059] FLAF p40, which the present inventors have firstly found, is anovel protein which hitherto never been reported and is one of importantsubunits constituting a molecule having the activity to activate felinelymphocytes to lead to destruction of viral infected cells. Furthermore,in accordance with the present invention, a whole amino acid sequence ofthe FLAF p35 molecule constituting the novel feline cytokine protein isdetermined to thereby reveal that the 19 amino acid residues attached atthe C terminal of the reported amino acid sequence of the feline IL12p35 is essential for the ability of activating lymphocytes. As mentionedhereinabove, the present invention dissolves the technical problemsassociated with the practical use of FLAF such as isolation of a genecoding for the active FLAF, construction of an expression vector,preparation of cells stably expressing FLAF, and purification of FLAF,and thus allows for production of FLAF as an agent for treating felineviral infectious diseases on an industrial scale.

[0060] Since FLAF of the present invention is a nonspecific factor whichis not specific to any particular virus, it can be applied to any virusin addition to herpes virus. Candidates for such a target virus include,for example, feline calicivirus, feline panleukopenia virus, felineleukemia virus, feline immunodeficiency virus, and the like. FLAF of thepresent invention can widely be applied to any immune reaction mediatedby histocompatibility class I antigen since it activates CTL. As such,FLAF of the present invention is expected to exhibit anti-tumoractivity.

[0061] The present invention is explained in more detail by way, of thefollowing Examples but it should not be construed to be limited thereto.In the following Examples, the reagents manufactured by Wako PureChemical Industries, Ltd., Takara Shuzo K. K., Toyobo K. K., GIBCO BRLor New England BioLabs were used unless otherwise mentioned.

EXAMPLE 1

[0062] Feline Lymphocyte Activating Factor

[0063] (1) Measurement of Feline Lymphocyte Activating Factor in CultureSupernatant of Feline Splenocytes

[0064] A human CTL enhancing activity against HSV-infected human cellswas measured in the manner described hereinbelow. For human PBMC, PBMCfrom subjects tested positive for anti-HSV antibody was used andselected from Buffy Coat available from the Japanese Red Cross Society,Blood Center. Target cells were prepared as described below.

[0065] Human PBMC at a concentration of 1×10⁶/ml are cultured in RPMImedium (Nissui Seiyaku K. K.) containing 10% fetal calf serum (FCS)under the conditions of 37° C. and 5% CO₂ for 3 days. On Day 3, to theculture is added 5 μg/ml of phytohemagglutinin and culture is continuedfor additional four days. On Day 7 after initiation of culture, thelymphocytes are harvested by centrifugation (1,500 rpm, 5 minutes) andsuspended in HSV (10⁸ TCID₅₀) solution. After incubation at 37° C. for 1hour, to the suspension is added RPMI and the mixture is centrifuged toremove HSV. This procedure is repeated twice and human PBMC is finallylabelled with sodium ⁵¹Cr (Amersham, CJS-11) in accordance with Gorse etal. (J. Clinical Microbiology Vol. 28, 2539-2550 (1990)) to give targetcells.

[0066] Effector cells were prepared as described below. Anti-HSVantibody positive PBMC was suspended in part (1×10⁷) in HSV solution andincubated at 37° C. for 1 hour. To the suspension was added RPMI and themixture was centrifuged. This procedure was repeated twice to removeHSV. To the resulting lymphocytes were added untreated lymphocytes 9×10⁷suspended in RPMI medium containing 10% FCS. Thereto was added 1/100volume of concentrated culture supernatant of feline spleno-cytes orculture supernatant of feline kidney cells as a negative control. Thelymphocytes were cultured under the conditions of 37 °C. and 5% CO₂ for7 days. On Day 7, the culture was Ficoll-centrifuged with Ficoll-PaquePlus (Pharmacia) at 1,500 rpm for 30 minutes to isolate the living cellswhich were used as effector cells.

[0067] CTL assay was initiated by mixing the target cells with theeffector cells. The target cells and the effector cells (1:50, 1:25;total 200 μl) were mixed on 96 well microtiter plate (Coning) andincubated under the conditions of 37° C. and 5% CO₂ for 6 hours. Sixhours later, ⁵¹Cr in the culture medium of each well was measured with γcounter (Aroca, ARC-360) on harvesting kit (Dainippon Seiyaku K. K.). Arelease percent of specific ⁵¹Cr was calculated as follows:

(n−min) / (Max−min)×100

[0068] wherein “n” means ⁵¹Cr count released in each sample, “min” means⁵¹Cr count released in those wells where target cells alone were addedwithout effector cells, and “Max” means ⁵¹Cr count released in thosewells where 10 μl of 10% Triton X-100 without effector cells so that⁵¹Cr is completely released. As is clear from the results obtained inthis CTL assay, the culture supernatant of feline splenocytessignificantly enhanced the CTL activity as compared with the negativecontrol, feline kidney cells culture supernatant (FIG. 1). This assayrevealed the CTL enhancing activity with high reproducibility. Theobtained results proved that the concentrated culture supernatant offeline splenocytes exhibits the CTL enhancing activity to human PBMC,suggesting the presence of a lymphocyte activating factor in theconcentrated culture supernatant of feline splenocytes.

[0069] (2) Measurement of activity to induce γ interferon to human PBMCin culture supernatant of feline splenocytes

[0070] As an index of lymphocyte activation, the γ interferon inducingactivity is also known in addition to the CTL activation. Thus, theactivity to induce γ interferon to human PBMC was measured in culturesupernatant of feline splenocytes. The activity was measured asdescribed below.

[0071] Normal adults were bled with heparin and blood wasFicoll-centrifuged (1,500 rpm, 30 minutes) to prepared PBMC, which wasfilled in a 96 well plate at 1×10⁶/well. To the plate was added 1/10volume of culture supernatant of feline splenocytes and incubated underthe conditions of 37° C. and 5% CO₂ for 16 hours. As a negative control,culture supernatant of CRFK cells, known feline kidney cell line, wasused. Sixteen hours later, the culture supernatant was harvested andhuman γ interferon in the culture supernatant was measured with EIA kit(Ohtsuka Kagaku Seiyaku K. K.). As a result, it was found that γinterferon was detected only in the wells where culture supernatant ofsplenocytes was added to prove that the culture supernatant of felinesplenocytes has the activity to induce γ interferon to human PBMC (FIG.2).

[0072] (3) Partial purification of a lymphocyte activating factor inculture supernatant of feline splenocytes

[0073] In order to determine whether these two activities wereattributable to a single factor, the concentrated culture supernatant offeline splenocytes was fractionated with an anion exchangechromatography to determine whether fractions having each of theseactivities were overlapped.

[0074] Culture supernatant (concentrated by 50 folds; 5 ml) of felinesplenocytes was dialyzed against 20 mM Tris-HCl buffer (pH 8.0)containing 50 mM NaCl and then applied to Mono-Q (Pharmacia, HR5/5)column equilibrated with the same buffer at a flow rate of 0.5 ml/min.The column was washed with 5 ml of the above buffer and eluted by a saltgradient with 10 ml of 50 mM -500 mM NaCl/20 mM Tris-HCl (pH 8.0) at aflow rate of 0.5 ml/min to give each 1 ml of fractions. Each offractions was dialyzed against PBS and then filtered with 0.22 μmfilter.

[0075] The CTL enhancing activity was measured for each of fractions bythe above system using human PBMC and HSV (target cells : effector cells=1 : 25). As a result, it was shown that the CTL enhancing activity wasexhibited by those fractions eluted at around 200 mM NaCl. The activityto induce γ interferon to human PBMC was also measured for each offractions. As a result, it was found that the γ interferon inducingactivity was also exhibited by those fractions eluted at around 200 mMNaCl like in the CTL enhancing activity. As shown in FIG. 3, a patternof eluted fractions was well matched for the CTL enhancing activity andthe γ interferon inducing activity, and thus both activities wereattributable to a single molecule. This feline factor having theseactivities was referred to as a feline lymphocyte activating factor andwas further purified. In the purification procedures described below,the γ interferon inducing activity was measured as an index activity.

EXAMPLE 2

[0076] Purification of Feline Lymphocyte Activating Factor

[0077] (1) Anion Exchange Chromatography

[0078] Culture supernatant (concentrated by 50 folds; 200 ml) of felinesplenocytes was dialyzed against 20 mM Tris-HCl buffer (pH 8.0)containing 50 mM NaCl and then applied at a flow rate of 2 ml/min toMono-Q (Pharmacia, HR10/10) column equilibrated with the same buffer.The column was washed with 50 ml of the above buffer and eluted at aflow rate of 2 ml/min by a salt gradient with 200 ml of 50 mM -500 mMNaCl/20 mM Tris-HCl (pH 8.0). A portion of fractions was used to measurethe γ interferon inducing activity. FIG. 4 shows the elution profile.The fractions having the activity were pooled and were dialyzed against20 mM citrate buffer (pH 6.0) containing 100 mM NaCl.

[0079] (2) Cation Exchange Chromatography

[0080] The dialysate obtained by the anion exchange chromatography wasapplied at a flow rate of 1 ml/min to SP-Toyopearl (Toso) column (1×8cm) equilibrated with 20 mM citrate buffer (pH 6.0) containing 100 mMNaCl. The column was washed with 50 ml of the above buffer and eluted bya salt gradient with 100 ml of 100 mM -800 mM NaCl/20 mM citrate (pH6.0) at a flow rate of 1 ml/min. A portion of fractions was used tomeasure the γ interferon inducing activity. FIG. 5 shows the elutionprofile. The fractions having the activity were pooled and were dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 200 mM NaCl.

[0081] (3) Heparin Column

[0082] The dialysate obtained by the cation exchange chromatography wasapplied at a flow rate of 0.5 ml/min to HiTrap-Heparin (Pharmacia) whichwas washed with 20 ml of 10 mM phosphate buffer (pH 7.0) containing 200mM NaCl and eluted by a salt gradient with 200 mM -1000 mM NaCl/10 mMphosphate buffer (pH 7.0). A portion of fractions was used to measurethe γ interferon inducing activity. FIG. 6 shows the elution profile.Finally, 5 ml fractions having the activity were obtained andconcentrated to 2 ml with Centricon-10 (Grace Japan).

[0083] (4) Gel Filtration

[0084] The concentrate obtained by the heparin column was applied to Gelfiltration column HiLoad 16/60 (Pharmacia) equilibrated with 10 mMphosphate buffer (pH 7.0) containing 100 mM NaCl and fractionated at aflow rate of 0.5 ml/min. Markers of Gel filtration, Bio Rad, were usedas a molecular weight standard. The γ interferon inducing activity wasmeasured for each fraction to reveal a peak activity at fractions ofmolecular weight around 60,000 to 80,000 (FIG. 7).

[0085] As shown in Table-1, the feline lymphocyte activating factorcould be purified up to 13% of an activity yield and a higher specificactivity by 2350 folds with an index of the γ interferon inducingactivity in accordance with the above 4 step purification procedures.Activity Total Specific activity Pruification (Induced γ-IFN) OD280Amount activity Yield Induced γ-IFN/OD280 degree Feline splenocyte 0.2ng/ml 0.12 6000 ml 1200 100% 1.7 1 culture Mono Q   9 0.63  50 450  3814.3 8.4 SP-Toyopearl  58 0.05   5 290  24 1160 680 Hi Trap Heparin  750.03   3 225  19 2500 1470 Hi Load  80 0.02   2 160  13 4000 2350

EXAMPLE 3

[0086] N Terminal Amino Acid Analysis of Feline Lymphocyte ActivatingFactor

[0087] Among the fractions fractionated by the gel filtration, thosefractions exhibiting the γ interferon inducing activity were subjectedto sodium dodecyl sulfate electrophoresis (SDS-PAGE) in the presence of2-mercaptoethanol to prove a unique main band of a molecular weight ofabout 40,000 associated with the γ interferon inducing activity (FIG.8). A minor band of a molecular weight of about 35,000 was alsoconfirmed. Firstly, the protein having a molecular weight of about40,000 was referred to as “FLAF p40”, and an amino acid sequence of itsN terminal was determined as described below.

[0088] The fractions with the activity were concentrated and, aftertreatment with 2-mercaptoethanol, subjected to SDS-PAGE with 10%polyacrylamide gel. After completion of electrophoresis, the gel wasimmersed into a transfer buffer (10 mMN-cyclohexyl-3-aminopropansulfonic acid, 10% methanol, pH 11) for 5minutes, overlaid to PVDF membrane (Immovilon: Millipore), which haspreviously been immersed successively into 100% methanol and thetransfer buffer, and the protein was transferred with TRANS-BLOTCELL(Bio Rad) at 160 mA for 16 hours. The PVDF membrane after transfer waswashed with water, stained with 0.1% amide black (40% methanol, 1%acetic acid) for 1 minute and washed with distilled water.

[0089] The stained band of a molecular weight of 40,000 was excised andthe membrane segment was analyzed with 477A Protein Sequencer (AppliedBio Systems). The N terminal amino acid sequence of 20 amino acidresidues was determined as follows:

[0090]Ile-Trp-Glu-Leu-Glu-Lys-Asn-Val-Tyr-Val-Val-Glu-Leu-Asp-Trp-His-Pro-Asp-Ala-Pro

[0091] as shown in SEQ ID NO: 11. Homology search was carried out forthis sequence with GENETYX software to prove that this protein hadhomology with human and mouse IL12 p40. However, the protein of thissequence has never been reported in cats.

EXAMPLE 4

[0092] Cloning of FLAF p40 Gene

[0093] In order to confirm that FLAF p40 actually possesses the CTLenhancing activity or the γ interferon inducing activity, an FLAF p40gene was firstly isolated from activated feline splenocyte cDNA library.The cDNA library was prepared as described below.

[0094] First, a whole RNA was prepared from feline splenocytes (culturedin the presence of 0.01% PWM for 18 hours, 3×10⁸) with ISOGEN reagent(Nippon Gene) and its protocol. Poly A+RNA was prepared from the wholeRNA with poly(A) quick mRNA isolation kit (STRATAGENE). Felinesplenocyte cDNA library was prepared from the poly A+RNA with Uni-ZAP XRvector kit (STRATAGENE) and its protocol. As a probe for screening, thesequence:

[0095] Ile-Trp-Glu-Leu-Glu-Lys-Asn-Val-Tyr-Val-Val-Glu-Leu

[0096] was selected from the N terminal amino acid sequence of 20 aminoacid residues of SEQ ID NO: 11 which was determined by analysis of the Nterminal amino acid sequence of FLAF p40, and an oligonucleotide:

[0097] ATCTGGGA(G,A)CT(G,C)GA(G,A)AA(G,A)AACGT(G,C)TACGT(G,C)GT(G,C)GA(G,A)CT

[0098] as shown in SEQ ID NO: 12 was designed and synthesized byemploying those codons used in cats at high frequency. Using 6 thisprobe, screening was carried out for 3×10⁶ plaques as described inMolecular cloning, A Laboratory Manual 2nd Ed., 12.30, Cold SpringHarbor Laboratory Press, N.Y., 1989 to give 2 positive plaques.

[0099] A nucleotide sequence of these cDNAs was determined to prove thatclone No. 12 had a nucleotide sequence coding for the amino acidsequence which is completely identical to the N terminal sequence ofFLAF p40. When the same frame as the N terminal sequence of FLAF p40 wasaligned to the nucleotide sequence of clone No. 12, it was revealed thatan open reading frame had an insert DNA of 500 bp in length and notermination codon. Thus, it was estimated that this cDNA does not encodea full length of FLAF p40. E. coli 92314 wherein plasmid (pFLAF12)comprising this clone No. 12 is incorporated has been deposited by theapplicant in accordance with the Budapest Treaty under accession numberFERM BP-5877 at National Institute of Bioscience and Human-TechnologyAgency of Industrial Science and Technology, 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, 305, Japan, on Mar. 14, 1997.

[0100] In order to obtain a clone of cDNA comprising a full length ofFLAF p40, feline splenocyte cDNA library was screened with a probeprepared on the basis of the insert region of the clone No. 12. Plaques(1×10⁶) were screened to give 1 positive clone (clone No. 25). Anucleotide sequence of the insert region of this clone No. 25 wasdetermined to prove that about 30 bp at the 5′ terminal of this clonewas overlapped with about 30 bp of the 3′ terminal of the clone No. 12and the 3′ terminal of the clone No. 25 comprises termination codon andpoly A. E. coli 92315 wherein plasmid (pFLAF25) comprising this cloneNo. 25 is incorporated has been deposited by the applicant in accordancewith the Budapest Treaty under accession number FERM BP-5878 at NationalInstitute of Bioscience and Human-Technology Agency of IndustrialScience and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,305, Japan, on Mar. 14, 1997. A combined nucleotide sequence of theclones No. 12 and No. 25 is shown as SEQ ID NO: 5. This nucleotidesequence revealed that FLAF p40 protein comprises a signal peptide,consisting of 22 amino acids, and 307 amino acid residues. Homologysearch for this sequence showed that this protein is a novel felineprotein.

EXAMPLE 5

[0101] Expression of FLAF p40 Gene

[0102] For expression of cDNA coding for FLAF p40 in an animal cell, itwas necessary to link the two clones obtained above together. However,since the overlapped region of these clones was quite short, i.e. onlyabout 30 bp, linkage of these clones by cleavage with a suitablerestriction enzyme and subsequent linkage was considered to bedifficult. Thus, by changing the codon GTG for No. 160 Val (thenumbering of an amino acid residue was in accord with SEQ ID NO: 1)contained in both clones into GTC (Val) thereby to introduce SalI siteinto this portion, the two clones were linked together in the mannerdescribed below.

[0103] In order to introduce SalI site into the 3′ terminal of the cloneNo. 12 and the 5′ terminal of the clone No. 25, polymerase chainreaction (PCR) was carried out with each clone being a template. PCRreaction was carried out with Amplitaq DNA polymerase (Takara Shuzo) for35 cycles, each cycle consisting of 95° C. for 1 minute, 60° C. for 2minutes and 72° C. for 2 minutes, with T3 primer of pBluescript IISK+vector and a primer of the sequence:

[0104] GGGGTACCGTCGACTCTGACCTTCTCTGCTGA

[0105] as shown in SEQ ID NO: 13 for the clone No. 12 and with T7 primerof pBluescript II SK+vector and a primer of the sequence:

[0106] GCTCTAGAAAGCTTGAATTCGTCGACAACAGGGATTATAAGAAG

[0107] as shown in SEQ ID NO: 14 for the clone No. 25.

[0108] The PCR products of each of clones 12 and 25 were digested withBglII-KpnI and XbaI-XcmI enzymes, respectively, and the resulting DNAsegments were ligated to pBluescript II SK+plasmids where each clonedigested with the same set of restriction enzymes was purified, tointroduce SalI site into each of the clone Nos. 12 and 25 on thepBluescript II SK+plasmid.

[0109] The substituted regions were sequenced with Dye Primer CycleSequencing Kit and Model 1373A DNA sequencer (Applied Bio Systems) toconfirm no error in the PCR reaction. Then, the clone 12 having SalIsite was digested with EcoRI-SalI to prepare a 5′ terminal DNA segmentof FLAF p40, which was then incorporated into the EcoRI-SalI site of theclone No. 25 plasmid having SalI site to link these two DNA segmentstogether to construct a full length p40 cDNA.

[0110] The thus obtained full length cDNA of FLAB p40 was incorporatedinto a suitable expression vector for an animal cell and an expressionwas carried out. pCAGn-mcs polyA (FIG. 10) was used as an expressionvector. pCAGn-mcs polyA vector contains human cytomegalovirus enhancerand chicken β actin promoter as an expression control region and rabbitβ globin splicing signal. After a SalI-EcoRI fragment of CAG enhancerpromoter (H. Niwa et al., Gene, 108, 193 (1991)) was blunt-ended andinserted into the EcoRV site of pBluescript II SK+vector, the terminalswere converted to EcoRI-HindIII site and then inserted into theEcoRI-HindIII site of pUC18 vector. An EcoRI-BamHI fragment was preparedfrom this plasmid and inserted into the EcoRI-BamHI site of pSV2neovector where HindIII site is deprived (P. J. Southern et al., J. Mol.Appl. Genet., 1, 327 (1982)), to which BamHI site was further inserted aDNA fragment containing a polyA addition signal of BamHI-BglII derivedfrom pCB25γSD/BcBg plasmid (Japanese patent publication No. 3-201986) toconstruct pCAGn-mcs polyA vector.

[0111] The plasmid containing the full length cDNA of FLAF p40 wasdigested with a restriction enzyme HindIII-XhoI to cleave the FLAF p40cDNA, which was inserted into the HindIII-SalI site of pCAGn-mcs polyAto construct a FLAF p40 expression vector. The obtained FLAF p40expression plasmid (CAGfp40) was introduced into COS7 cells withLipofect Ace reagent for transient expression.

[0112] The day (20 hours) before transfection, COS7 cells were plated ona 6 well plate at 2×10⁵ cells/well. On the next day, Lipofect Acereagent was mixed with the expression plasmid (1 μg of cAGf40 per well)and the mixture was left to stand at room temperature for 15 minutes andthen added to the COS7 cells, followed by addition of serum free mediumOpti-MEM (0.8 ml). On the next day, 2 ml of serum free medium (ASF104;Ajinomoto K. K.) was added and culture was continued for additional 2days and then culture supernatant was recovered. A portion of thesupernatant was subjected to Western blot as described in Example 11 todetect expression products which reacted with polyclonal antibodieswhich recognize FLAF p40. As a result, bands corresponding to FLAF p40reactive with anti-FLAF p40 antibody (MW about 40,000) and to ahomologous dimer thereof (MW about 80,000) were detected (FIG. 11).However, the culture supernatant exhibited no activity to induce γinterferon against human PBMC like the culture supernatant in the wellwhere pCAGn-mcs polyA was used for transfection as a negative control(FIG. 12). That is, it was revealed that the γ interferon inducingactivity exhibited by the fractions of molecular weight of around 60,000to 80,000 during purification by gel filtration of the feline lymphocyteactivating factor was not attributable to the homologous dimer of FLAFp40.

[0113] However, the homologous dimer of FLAF p40 of the presentinvention is expected to be an antagonist to the feline cytokine proteinto show a feline cytokine protein. inhibiting activity like thehomologous dimer of mouse IL12 β chain (p40) as reported by Germann etal., Immunology Today Vol. 16, p500-501 (1995).

EXAMPLE 6

[0114] Cloning of Feline IL12 p35 Gene

[0115] FLAF p40 isolated herein alone did not show the γ interferoninducing activity. Since the γ interferon inducing activity exhibitedduring purification by gel filtration of the feline lymphocyteactivating factor was not attributable to the homologous dimer of FLAFp40, it is possibly suggested that the activity is attributable to acomplex formed between the FLAF p40 molecule and a distinct moleculehaving a similar molecular weight to that of FLAF p40. In view of its γinterferon inducing activity and molecular weight, the feline lymphocyteactivating factor of the present invention may be homologous to human ormouse IL12 (heterologous dimer of p40 beta chain and p35 alpha chain).In this context, it is possible that the feline IL12 p35 with amolecular weight 35,000 as reported by Bush et al. is a partner of theFLAF p40 molecule of the present invention. Thus, a feline IL12 p35 genecoding for an amino acid sequence of feline IL12 p35 was cloned andrelation between feline IL12 p35 and FLAF p40 was investigated.

[0116] Based on the N terminal and C terminal amino acid sequences ofthe feline IL12 p35 as reported by Bush et al., there were prepared asynthetic DNA (1) of the nucleotide sequence:

[0117] ATGTGCCC(A,G,C,T)CC(A,G,C,T)CT(A,G,C,T)TG(T,C)CT(A,G,C,T)

[0118] as shown in SEQ ID NO: 15 and a synthetic DNA (2) of thenucleotide sequence:

[0119] ATGTGCCC(A,G,C,T)CC(A,G,C,T)TT(A,G)TG(T,C)TT(A,G)

[0120] as shown in SEQ ID NO: 16 as a PCR primer corresponding to the Nterminal sequence:

[0121] Met-Cys-Pro-Pro-Leu-Cys-Leu

[0122] and, a synthetic DNA (3) of the nucleotide sequence:

[0123] (G,A)TG(A,G,C,T)AG(A,G,C,T)AG(A,G,T)ATGCA(A,G,C,T)AGCTT

[0124] as shown in SEQ ID NO: 17 and a synthetic DNA (4) of thenucleotide sequence:

[0125] (A,G)TG(C,T)AA(C,T)AA(A,G,T)ATGCA(C,T)AACTT

[0126] as shown in SEQ ID NO: 18 as a PCR primer corresponding to the Cterminal sequence:

[0127] Lys-Leu-Cys-Ile-Leu-Leu-His.

[0128] By combining these two N terminal primers and the two C terminalprimers, each of 4 primer sets, (1)+(3), (1)+(4), (2)+(3), or (2)+(4),was used to conduct PCR with cDNA from activated feline splenocytes as atemplate. PCR reaction was carried out with Amplitaq DNA polymerase(Takara Shuzo) for 35 cycles, each cycle consisting of 95° C. for 1minute, 60° C. for 2 minutes and 72° C. for 2 minutes. The obtained PCRproducts were analyzed on 1% agarose gel. An amplified fragment of anexpected size (600 bp) from the primer set (1) +(3) was detected. TheDNA segment was excised from the gel and, using TA Cloning Kit(Invitrogen), cloned into pCRII vector contained in the kit.

[0129] In order to confirm that this DNA segment actually codes for theamino acid sequence of the feline IL12 p35 as reported by Bush et al.,primers corresponding to SP6 promoter and T7 promoter region containedin this vector were used to produce amplified fragments for sequencing.As a result, this DNA segment proved to code for the identical aminoacid sequence to the reported feline IL12 p35 ranging from the Nterminal methionine to the C terminal histidine. Thus, this DNA segmentwas used for expression as described below.

EXAMPLE 7

[0130] Co-expression of FLAF p40 and Feline IL12 p35 Genes The cDNA offeline IL12 p35 obtained in Example 6 was cleaved from pCRII vector withrestriction enzymes SacI and XhoI and the cleavage sites wereblunt-ended with T4 DNA polymerase. pCAGn-mcs polyA vector was cleavedwith a restriction enzyme SalI, the cleavage sites were blunt-ended withT4 DNA polymerase and the 5′ terminal was dephosphorylated withbacterial alkaline phosphatase. To the resulting vector was ligated theabove cDNA fragment of feline IL12 p35 with T4 ligase to construct anexpression vector for feline IL12 p35 (CAGfIL12p35).

[0131] The thus prepared expression vector for feline IL12 p35(CAGfIL12p35) alone (1 μg) or in combination with CAGfp40 (each 0.5 μg)were used to transfect COS7 cells using Lipofect Ace reagent and theprocedures as described in Example 6. Each culture supernatant wasmeasured for their activity to induce γ interferon to human PBMC butboth failed to detect the activity (FIG. 12). That is, it was shown thata combination of the protein having the amino acid sequence of felineIL12 p35 as reported and the FLAF p40 protein did not exhibit the γinterferon inducing activity. The activity could not also be detectedwith cDNA of feline IL12 p35 alone. From this, it was possible thateither (1) another molecule other than IL12 p35 is necessary for theactivity, or (2) there may not be a full length amino acid sequence offeline IL12 p35 as reported by Bush et al.

[0132] As describe above, SDS-PAGE analysis in the presence of2-mercaptoethanol of the gel filtration fractions having the γinterferon inducing activity revealed a band of molecular weight 35,000as well as the unique main band of molecular weight 40,000 (FIG. 8).This protein of molecular weight 35,000 was referred to as “FLAF p35”and the N terminal was sequenced with 477A Protein Sequencer (AppliedBio Systems) as described for FLAF p40. The N terminal amino acidsequence of 10 amino acid residues was determined to be:

[0133] Arg-Asn-Leu-Pro-Thr-Pro-Thr-Pro-Ser-Pro

[0134] as shown in SEQ ID NO: 19. In comparison with the N terminalamino acid sequence of feline IL12 p35 as reported by Bush et al., thisamino acid sequence was completely identical. In this context, weconsidered that the feline IL12 p35 as reported by Bush et al. isdefective for exerting the activity and thus cloned cDNA coding for theFLAF p35 molecule (FLAF p35 cDNA).

EXAMPLE 8

[0135] Cloning of FLAF p35 Gene

[0136] FLAF p35 cDNA was cloned as described below. First, a whole RNAwas prepared from feline splenocytes 3×10⁸ (cultured in the presence of0.01% PWM for 18 hours) in accordance with the protocol of ISOGENreagent (Nippon Gene). Poly A+RNA was prepared from the whole RNA withpolyA quick mRNA isolation kit (STRATAGENE). Feline splenocyte cDNAlibrary was prepared from the poly A+RNA in accordance with the protocolof Uni-ZAP XR vector kit (STRATAGENE).

[0137] The PCR product coding for the feline IL12 p35 as described abovewas used as a probe for screening 2×10⁶ plaques of the cDNA library asdescribed in Maniatis et al., Molecular cloning, A Laboratory Manual 2ndEd., 8.46, Cold Spring Harbor Laboratory Press, N.Y., 1989 to give 3positive plaques.

[0138] A whole nucleotide sequence of cDNA of these three plaques wasdetermined to prove that a single clone (clone No. 20) had the aminoacid sequence which was completely identical to the N terminal sequenceof IL12 p35 (nucleotide sequence of clone No. 20 is described in SEQ IDNO: 8). When an open reading frame was determined for the nucleotidesequence of clone No. 20, coding regions for 25 amino acid residues,possibly a signal sequence, and for 197 amino acid residues were found.In comparison of this sequence with the reported amino acid sequence offeline IL12 p35, there were found difference in an amino acid residue at2 sites and insertion of 3 amino acid residues within the signalsequence as well as 19 amino acid residues attached to the C terminal offeline IL12 p35 (FIG. 13). As such, it was shown that the proteinencoded by FLAF p35 cDNA is a protein having a partially distinct aminoacid sequence from that of feline IL12 p35. E. coli 92313 whereinplasmid (pFLAF20) comprising this clone No. 20 is incorporated has beendeposited by the applicant in accordance with the Budapest Treaty underaccession number FERM BP-5876 at National Institute of Bioscience andHuman-Technology Agency of Industrial Science and Technology, 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305, Japan, on Mar. 14, 1997.

EXAMPLE 9

[0139] Co-expression of FLAF p40 and FLAF p35

[0140] For expression of the obtained FLAF p35 cDNA in COS7 cells, theFLAF p35 cDNA was inserted into pCAGn-mcs polyA vector as described forFLAF p40 cDNA to construct an expression plasmid. First, the full lengthcDNA of FLAF p35 was cleaved from the clone No. 20 obtained herein withrestriction enzymes SacI and XhoI and the cleavage sites wereblunt-ended with T4 DNA polymerase. The resulting cleaved DNA fragmentwas inserted into the SalI site of pCAGn-mcs polyA (after blunt-endingand dephosphorylation) to construct an expression plasmid. The thusprepared expression vector for FLAB p35 and the expression vector forFLAF p40 as described above were used to co-transfect COS7 cells usingLipofect Ace reagent as described in Example 6. The culture supernatantwas recovered and measured for its activity to induce γ interferon tohuman PBMC, and as a result, the activity was detected in the culturesupernatant (FIG. 12). Furthermore, the culture supernatant was alsomeasured for its CTL enhancing activity in the human herpes virus systemas described in Example 1, and as a result, the same CTL enhancingactivity as found by the present inventors in the culture supernatant offeline splenocyte was detected (FIG. 14).

[0141] Thus, it was found that the FLAF p35 and p40 of the presentinvention in the form of a heterologous dimer exhibit the γ interferoninducing activity and the CTL enhancing activity, indicating that eachcDNA for the FLAF p35 and p40 together encode a protein having the CTLenhancing activity as found by the present inventors in the culturesupernatant of feline splenocyte. Furthermore, feline IL12 p35 aspreviously reported in the literatures could not exhibit the lymphocyteactivating capacity even if it is co-expressed with the FLAF p40 foundby the present inventors. This suggests that the FLAF activity requiresnot only the amino acid sequence of feline IL12 p35 as reported but alsothe additional sequence of 19 amino acid residues attached to the Cterminal of feline IL12 p35 as mentioned above. Thus, the presentinventors have found that said additional sequence of 19 amino acidresidues is essential for exerting the CTL enhancing activity and the γinterferon inducing activity.

EXAMPLE 10

[0142] Preparation of Transformants Which Stably Produce FLAF

[0143] The results obtained with COS7 cells revealed that the felinelymphocyte activating factor is a heterologous dimer (FLAF) of peptidesencoded by FLAF p35 and p40 cDNAs, respectively. However, since theexpression with COS7 cells is merely transient, it is necessary toprepare cells which can stably produce FLAF for the purpose of preparingFLAF as a medicament. Thus, a stable transformant was prepared withSFT34 cells (Patent, International publication No. WO94/12658) derivedfrom mouse myeloma as a host. The SFT34 cells can easily be suspended ina culture medium and quickly be adapted to a serum free culture medium.Each of FLAF p35 and p40 cDNAs was introduced into a CAG vector havingG418 gene (Neo gene) as a selective marker. The vectors incorporatingeither FLAF p35 or p40 cDNAs were simultaneously introduced into theSFT34 cells with Lipofect Ace reagent. The cells were cultured on 10%FCS/RPMI medium containing 1 mg/ml G418 for 2 weeks to give G418resistant SFT34 cells. The transformants were cloned by a limitingdilution procedure and those transformants which stably produced FLAFwere obtained with the use of an index of the γ interferon inducingactivity (FIG. 15). Culture supernatant (3 liters) of these clones wassubjected to the purification procedures as described above. As aresult, the obtained purified products exhibited the CTL enhancingactivity and the γ interferon inducing activity like the FLAF purifiedfrom feline splenocytes, and thus, it became possible to stably obtainFLAF.

EXAMPLE 11

[0144] Preparation of antibody against FLAF p40

[0145] A polyclonal antibody against FLAF p40 was prepared by injectingeach 1 ml of the FLAF p40 expression plasmid (CAGfp40) (1 mg/ml; Example5) intramuscularly into both rear legs of a rabbit four times at every 3weeks. Western blot was used to determine whether the obtained antibodyrecognized FLAF p40 (FIG. 16). First, purified FLAF was subjected toSDS-PAGE in the absence of 2-mercaptoethanol and then transferred toPVDF membrane as described in Example 3. After the membrane was maskedwith 50 mM Tris-HCl (pH 8.0) supplemented with 5% skim milk, 150 mMNaCl, and 0.05% Tween 20 (TBST), it was incubated with serum from therabbit administered with CAGfp40 diluted by 500 folds with TBST for 1hour and then washed with TBST. Subsequently, the membrane was reactedwith anti-rabbit IgG-HRP labeled antibody (Bio Rad) diluted by 2,000folds with TBST at room temperature for 1 hour and, after washing,stained with Konica Immunostaining HRP 1000 (Konica K. K.) kit. As aresult, it reacted with the FLAF p40 protein confirmed by the N terminalamino acid analysis. Furthermore, Western blot of the fractions obtainedduring purification of FLAF p40 with this antibody revealed that thefractions which reacted with the antibody overlapped with the fractionswhich exhibited the γ interferon inducing activity to ensure that theantibody can be used in the assay system of FLAF.

EXAMPLE 12

[0146] Preparation of Monoclonal Antibody Against Partial SyntheticPeptide of FLAF p35

[0147] The amino acid sequence of FLAF p35 was analyzed for itshydrophilic and hydrophobic regions in accordance with Hopp et al. (T.P. Hopp et al., Proc. Natl. Acad. Sci. (USA), Vol. 78, 3824-3828, 1981).For the hydrophilic region, two peptides were prepared with a peptidesynthesizer (Applied), each peptide having the sequence:

[0148]Thr-Ser-Glu-Glu-Ile-Asp-His-Glu-Asp-Ile-Thr-Lys-Asp-Lys-Thr-Ser-Thr-Val-Glu-Ala-Cys

[0149] as shown in SEQ ID NO: 20 which corresponds to the amino acidresidue Nos. 43 to 63 in the sequences of SEQ ID NOs: 3 and 4, and thesequence:

[0150] Ser-Ser-Leu-Glu-Glu-Pro-Asp-Phe-Tyr-Lys-Thr-Lys-Ile-Lys-Leu-Cys

[0151] as shown in SEQ ID NO: 21 which corresponds to the amino acidresidue Nos. 159 to 174 in the sequences of SEQ ID NOs: 3 and 4,respectively. Immunization with these two peptides is described below.

[0152] A group of gld mice of 6 weeks old (Nippon S L C K. K.) were usedfor immunization consisting of three intraperitoneal injections and oneintravenous injection of the peptides. A synthetic peptide-KLH conjugate100 μg was injected in the presence of complete Freund's adjuvant on Day0, in the presence of incomplete Freund's adjuvant on Days 14 and 28,and in the absence of adjuvant on Day 42. Three days after the finalimmunization, splenocytes were harvested by the conventional procedures.Fusion of the splenocytes with myeloma cells p3×63Ag-8U1 was conductedin accordance with Köhler and Milstein (Nature, 256, p495, 1975). A cellsuspension after cell fusion was placed on a 96 well plate at 200μl/well and the cells were cultured in an incubator containing 5% CO₂ at37° C. for 24 hours. HT medium (normal medium supplemented withhypoxanthine 1×10⁻⁴M and thymidine 1.5×10⁻³ M) was then added and thecells were cultured for additional 10 to 14 days. Among the culturesupernatant from the wells where the fused cells grew to form colonies,a desired hybridoma was selected by the screening procedure as describedbelow.

[0153] A desired hybridoma was selected by a combination of EIA andWestern blot.

[0154] A primary screening by EIA is first described. The peptidicantigens as prepared above were immobilized on a 96 well plate at 2μg/ml and the plate was masked with a 1% solution of bovine serumalbumin. The plate was added with the culture supernatant of thehybridomas obtained by cell fusion, incubated at 4° C. for 2 hours andwashed with 0.1% Tween 20/PBS three times. The plate was added with asolution of an anti-mouse immunoglobulin antibody labeled withperoxidase (Kappel; diluted by 5,000 folds; 100 μl), incubated at 4° C.for 1 hour and washed with 0.1% Tween 20/PBS three times. Subsequently,a TMBZ substrate solution was added to develop in the usual manner andan absorbance at 450 nm was measured. The thus selected wells whichreacted with the synthetic peptides were subjected to a secondaryscreening with Western blot.

[0155] The purified FLAF was subjected to SDS-PAGE in the absence of2-mercaptoethanol and then transferred to PVDF membrane as described inExample 11. The membrane was masked, added with the culture supernatantwhich tested positive in the EIA screening to react at room temperaturefor 1 hour and washed with TBST,. The membrane was then reacted with ananti-mouse IgG-HRP labeled antibody (Bio Rad; diluted by 2,000 folds) atroom temperature for 1 hour and, after washing, stained with KonicaImmunostaining HRP 1000 kit (Konica K. K.). As a result, a specificreaction with FLAF was observed for the antibodies produced by thehybridomas from 4 clones (F6-13, F6-15, F6-17 and F6-18) obtained byimmunization with the synthetic peptide of SEQ ID NO: 20 and from 3clones (F18-2, F18-4 and F18-18) obtained by immunization with thesynthetic peptide of SEQ ID NO: 21. Among these clones, the hybridomasF6-13 and F18-4 have been deposited in accordance with the BudapestTreaty under accession numbers FERM BP-5924 and FERM BP-5925,respectively, at National Institute of Bioscience and Human-TechnologyAgency of Industrial Science and Technology, 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, 305, Japan, on Apr. 18, 1997.

[0156] Western blot of the fractions obtained during purification ofFLAF with the culture supernatant from these 7 clones revealed that thefractions which reacted with these antibodies overlapped with thefractions which exhibited the γ interferon inducing activity to ensurethat these antibodies can be used in the assay system of FLAF.

[0157] Then, a gel with an immobilized antibody was prepared byimmobilizing the antibody (subtype IgG1) obtained from, for example, theclone F18-4 on a CNBr-activated Sepharose (Pharmacia) in accordance withthe protocol of Pharmacia. The gel (1 ml) was equilibrated with 10 mMphosphate (pH 7.0)-100 mM NaCl buffer. To the gel was applied a crudeFLAF (10 ml) dialyzed against the same buffer. After washing with thesame buffer, elution was carried out with 0.1 M glycine HCl buffer (pH3.0). As a result, a high purity of FLAF was detected in the elutedfractions to prove that the gel with the immobilized monoclonal antibodycan be used for purification of FLAF.

EXAMPLE 13

[0158] Effect of FLAF for Ameliorating Feline Herpes Virus (FHV)Infection in Cats

[0159] Conventional cats obtained from the Animal Control Center ofKumamoto Prefecture were tested for an anti-FHV antibody titer afterbleeding and those cats tested negative for the anti-FHV antibody titerwere used in this experiment. The cats weighing 1 to 2.5 kg werecompulsorily infected nasally with FHV-1-K1 strain at 105 TCID₅₀. Ondays 2, 4 and 6 after the compulsory infection, the purified FLAF at 16μg/kg was intravenously administered via the jugular vein. A controlgroup (2 animals) received a physiological saline by an intravenousadministration via the jugular vein with the same schedule. The catswere observed for their clinical conditions such as tears,conjunctivitis, rhinorrhea, sneeze, slaver and ulcer at the nose edgewere observed with the lapse of time and scored as 0 for no symptom, 1through 3 for symptoms of various severity. FIG. 17 shows cumulativescores obtained by observation with the lapse of time. As a result, theclinical conditions were remedied in the group administered with FLAF ascompared to the control group, showing that FLAF is effective fortreating FHV infectious diseases in vivo. In addition, afteradministration of FLAF, no side effect such as fever, diarrhea, vomitingor another shock-like symptom was observed. Thus, FLAF can be applied astreating agents effective against FHV infectious diseases with no sideeffect.

EXAMPLE 14

[0160] Enhancement of Recombinant FLAF Expression With Animal Cells as aHost by Modification of FLAF p35 cDNA

[0161] As described in Example 9, FLAF having the biological activitycan be obtained by co-transfecting animal cells with the expressionplasmids wherein either FLAF p40 or p35 cDNA was incorporated. However,this procedures can only provide FLAF at as low as several μg/l. Inorder to improve the production rate, the FLAF p35 cDNA was modified asdescribed below.

[0162] Step 1

[0163] First, using the plasmid pFLAF20 obtained in Example 8 as atemplate, PCR reaction was carried out with Amplitaq DNA polymerase(Takara Shuzo K. K.) for 35 cycles, each cycle consisting of 95° C. for1 minute, 60° C. for 2. minutes and 72° C. for 2 minutes, using asynthetic primer having the sequence of SEQ ID NO: 22 corresponding tothe amino acid sequence ranging from the amino acid residues No. −25 toNo. −3 as shown in SEQ ID NO: 3 and a synthetic primer having thesequence of SEQ ID NO: 23 corresponding to the 3′ poly A sequence ofFLAF p35 cDNA. The primer having the sequence of SEQ ID NO: 22 comprisesKozak sequence (J. Cell. Biol., Vol. 108, 229-233, 1989) which isreported to enhance the translation efficiency at its N terminal. Theobtained DNA fragments were digested with restriction enzymes SacI andXhoI and subcloned into the vector pBluescript which has previously beendigested with the same enzymes.

[0164] Step 2

[0165] For the insertion region of the plasmid obtained in Step 1, anucleotide sequence of a region coding for an amino acid was determinedto confirm that no mutation such as a frame shift occurred. Among theamino acid residues in the coding region, Nos. 64, 68 and 124 leucine(Leu) as shown in SEQ ID NO: 3 are coded by the codon TTA. Among sixcodons used for encoding Leu, TTA is the least used in an animal cell(Current Protocols in Molecular Biology, Supplement 12 A.1.9.). Thus,this codon used for Leu at the three positions was replaced with CTG.First, there were prepared a synthetic DNA as shown in SEQ ID NO: 24corresponding to the sequence in the vicinity of the restriction enzymesite EcoRV site and a synthetic DNA as shown in SEQ ID NO: 25corresponding to the sequence in the vicinity of the restriction enzymesite BamHI site. Using these two synthetic DNAs as a primer, PCRreaction was carried out using the DNA obtained in Step 1 as a templateunder the conditions as described in Step 1. The DNA fragment amplifiedby this reaction was subjected to an agarose gel electrophoresis toresolve impurities and recovered from the gel. The DNA fragment wasdigested with BamHI and EcoRV and the obtained BamHI-EcoRV fragment wasinserted into the BamHI-EcoRV site of the plasmid obtained in Step 1. Anucleotide sequence of the DNA fragment inserted in the plasmid wasdetermined to confirm that no mutation such as a frame shift occurred.

[0166] Then, the plasmid was digested with restriction enzymes SacI andBsmAI so that the plasmid was cleaved at the SacI site situated justupstream of Kozak sequence and at the BsmAI site situated justdownstream of the termination codon to produce a fragment of about 800bp comprising a full length of the coding region of FLAF p35. Theresulting fragment was blunt-ended with T4 DNA polymerase (a nucleotidesequence of this fragment is shown in SEQ ID NO: 26). This DNA fragmentwas inserted into the expression vector pCAGn-mcs, which has previouslybeen digested with SalI and blunt-ended with T4 DNA polymerase, toconstruct a novel FLAF p35 expression plasmid (referred to asCAGfp35V2).

[0167] Both CAGfp35V2 and CAGfp40 as described in Example 5 weresimultaneously introduced into SFT cells as described in Example 10 toprepare a stable transformant. Each 2 liter sample of this stabletransformant and the stable transformant obtained in Example 10 werecultured. Each culture supernatant was purified as described in Example2 and using the gel with the immobilized monoclonal antibody against thesynthetic peptide of FLAF p35 as described in Example 12. As a result,the culture supernatant obtained from the transformant with CAGfp35V2could provide purified FLAF at a higher amount by 200 folds than theculture supernatant obtained from the transformant with CAGfp35.

[0168] The purified FLAF was subjected to SDS-PAGE in the absence of2-mercaptoethanol to prove two bands of MW 75,000 (referred to as p75)and MW 80,000 (referred to as p80) as shown in FIG. 18. These bands wereanalyzed by Western blot as described in Example 2 wherein two membraneswere prepared, i.e. one using a monoclonal antibody against the partialsynthetic peptide of FLAF p35 produced by the clone F18-4 as describedin Example 12 as a primary antibody and another using the antibodyagainst FLAF p40 as described in Example 11. As a result, both p75 andp80 reacted with both antibodies to confirm that they are bothheterologous dimer of FLAF p35 and FLAF p40.

[0169] In order to investigate the biological activity of p75 and p80,the activity to induce γ interferon against human PBMC was measured asdescribed in Example 1. Resolution of p75 and p80 was carried out asdescribed below. The purified FLAF was subjected to SDS-PAGE in theabsence of 2-mercaptoethanol, the gel was stained with Kappa Stain Kit(Bio Rad) and each gel comprising the bands of p75 and p80 was excised.These gels were separately covered in a dialysis tube, thereto was added200 μl of 25 mM Tris, 192 mM glycine buffer, and electricity was chargedusing an electrophoretic bath filled with the same buffer at 50 voltsfor 1 hour. FLAF eluted out of the gel into the dialysis tube wasdialyzed against PBS. The obtained sample was tested for the activity toinduce γ interferon against human PBMC using PBS as a control.

[0170] As a result, both p75 and p80 exhibited the γ interferon inducingactivity as shown in FIG. 19.

Reference Example

[0171] Cloning of FLAF p35 and p40 cDNAs Using Human IL12 α Chain and βChain cDNAs as a Probe

[0172] It was confirmed that the FLAF p35 and p40 obtained herein had ahigh homology with human IL12 α chain and β chain as a result ofhomology search for their nucleotide sequence with the known data base(for example, GenBank, SWISS-PROT etc.). Cloning of FLAF p35 and FLAFp40 cDNAs is described in Examples 4 and 8, respectively. However, theseprocedures are not only quite cumbersome but also provide a positiveclone only at a quite low rate. Thus, in order to clone FLAF p35 and p40cDNAs more efficiently, human IL12 α chain and γ chain cDNAs were clonedby using PCR and, using these human IL12 cDNAs as a probe, the cDNAlibrary prepared as described in Example 4 was screened. As a result,one FLAF p35 cDNA and two FLAF p40 cDNAs were detected among 2×10⁶plaques. A nucleotide sequence of these clones was determined to becompletely identical to the sequences of SEQ ID NOs: 5 and 8. Thus, itwas found that FLAF p35 and FLAF p40 cDNAs could easily be cloned byusing human IL12 α chain and β chain cDNAs as a probe.

1 26 329 amino acids amino acid single linear protein 1 Met His Pro GlnGln Leu Val Ile Ala Trp Phe Tyr Leu Val Leu Leu 1 5 10 15 Ala Pro ProLeu Met Ala Ile Trp Glu Leu Glu Lys Asn Val Tyr Val 20 25 30 Val Glu LeuAsp Trp His Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys AsnThr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60 Ser Ser GluVal Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu PheAla Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu SerHis Ser Phe Leu Leu Ile His Lys Lys Glu Asp Gly Ile Trp 100 105 110 SerThr Asp Ile Leu Arg Glu Gln Lys Glu Ser Lys Asn Lys Ile Phe 115 120 125Leu Lys Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135140 Leu Thr Ala Ile Ser Thr Asp Leu Lys Phe Thr Val Lys Ser Ser Arg 145150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr LeuSer 165 170 175 Ala Glu Lys Val Arg Val Asp Asn Arg Asp Tyr Lys Lys TyrThr Val 180 185 190 Glu Cys Gln Glu Gly Ser Ala Cys Pro Ala Ala Glu GluSer Leu Pro 195 200 205 Ile Glu Val Val Val Asp Ala Ile His Lys Leu LysTyr Glu Asn Tyr 210 215 220 Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile LysPro Asp Pro Pro Lys 225 230 235 240 Asn Leu Gln Leu Lys Pro Leu Lys AsnSer Arg His Val Glu Val Ser 245 250 255 Trp Glu Tyr Pro Asp Thr Trp SerThr Pro His Ser Tyr Phe Ser Leu 260 265 270 Thr Phe Gly Val Gln Val GlnGly Lys Asn Asn Arg Glu Lys Lys Asp 275 280 285 Arg Leu Ser Val Asp LysThr Ser Ala Lys Val Val Cys His Lys Asp 290 295 300 Ala Lys Ile Arg ValGln Ala Arg Asp Arg Tyr Tyr Ser Ser Ser Trp 305 310 315 320 Ser Asn TrpAla Ser Val Ser Cys Ser 325 307 amino acids amino acid single linearprotein 2 Ile Trp Glu Leu Glu Lys Asn Val Tyr Val Val Glu Leu Asp TrpHis 1 5 10 15 Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asn ThrPro Glu 20 25 30 Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Ser Ser Glu ValLeu Gly 35 40 45 Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Ala AspAla Gly 50 55 60 Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His SerPhe Leu 65 70 75 80 Leu Ile His Lys Lys Glu Asp Gly Ile Trp Ser Thr AspIle Leu Arg 85 90 95 Glu Gln Lys Glu Ser Lys Asn Lys Ile Phe Leu Lys CysGlu Ala Lys 100 105 110 Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu ThrAla Ile Ser Thr 115 120 125 Asp Leu Lys Phe Thr Val Lys Ser Ser Arg GlySer Ser Asp Pro Gln 130 135 140 Gly Val Thr Cys Gly Ala Ala Thr Leu SerAla Glu Lys Val Arg Val 145 150 155 160 Asp Asn Arg Asp Tyr Lys Lys TyrThr Val Glu Cys Gln Glu Gly Ser 165 170 175 Ala Cys Pro Ala Ala Glu GluSer Leu Pro Ile Glu Val Val Val Asp 180 185 190 Ala Ile His Lys Leu LysTyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile 195 200 205 Arg Asp Ile Ile LysPro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro 210 215 220 Leu Lys Asn SerArg His Val Glu Val Ser Trp Glu Tyr Pro Asp Thr 225 230 235 240 Trp SerThr Pro His Ser Tyr Phe Ser Leu Thr Phe Gly Val Gln Val 245 250 255 GlnGly Lys Asn Asn Arg Glu Lys Lys Asp Arg Leu Ser Val Asp Lys 260 265 270Thr Ser Ala Lys Val Val Cys His Lys Asp Ala Lys Ile Arg Val Gln 275 280285 Ala Arg Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Asn Trp Ala Ser Val 290295 300 Ser Cys Ser 305 222 amino acids amino acid single linear protein3 Met Cys Pro Pro Arg Gly Leu Leu Leu Val Thr Ile Leu Val Leu Leu 1 5 1015 Asn His Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Thr Pro Thr 20 2530 Pro Ser Pro Gly Met Phe Gln Cys Leu Asn His Ser Gln Thr Leu Leu 35 4045 Arg Ala Ile Ser Asn Thr Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe 50 5560 Tyr Ser Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp 65 7075 80 Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Met Asn 8590 95 Glu Ser Cys Leu Ala Ser Arg Glu Ile Ser Leu Ile Thr Asn Gly Ser100 105 110 Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Thr Thr Leu Cys LeuSer 115 120 125 Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe LysAla Met 130 135 140 Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile PheLeu Asp Gln 145 150 155 160 Asn Met Leu Thr Ala Ile Asp Glu Leu Leu GlnAla Leu Asn Val Asn 165 170 175 Ser Val Thr Val Pro Gln Asn Ser Ser LeuGlu Glu Pro Asp Phe Tyr 180 185 190 Lys Thr Lys Ile Lys Leu Cys Ile LeuLeu His Ala Phe Arg Ile Arg 195 200 205 Ala Val Thr Ile Asn Arg Met MetSer Tyr Leu Asn Ser Ser 210 215 220 197 amino acids amino acid singlelinear protein 4 Arg Asn Leu Pro Thr Pro Thr Pro Ser Pro Gly Met Phe GlnCys Leu 1 5 10 15 Asn His Ser Gln Thr Leu Leu Arg Ala Ile Ser Asn ThrLeu Gln Lys 20 25 30 Ala Arg Gln Thr Leu Glu Phe Tyr Ser Cys Thr Ser GluGlu Ile Asp 35 40 45 His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val GluAla Cys Leu 50 55 60 Pro Leu Glu Leu Thr Met Asn Glu Ser Cys Leu Ala SerArg Glu Ile 65 70 75 80 Ser Leu Ile Thr Asn Gly Ser Cys Leu Ala Ser ArgLys Thr Ser Phe 85 90 95 Met Thr Thr Leu Cys Leu Ser Ser Ile Tyr Glu AspLeu Lys Met Tyr 100 105 110 Gln Val Glu Phe Lys Ala Met Asn Ala Lys LeuLeu Met Asp Pro Lys 115 120 125 Arg Gln Ile Phe Leu Asp Gln Asn Met LeuThr Ala Ile Asp Glu Leu 130 135 140 Leu Gln Ala Leu Asn Val Asn Ser ValThr Val Pro Gln Asn Ser Ser 145 150 155 160 Leu Glu Glu Pro Asp Phe TyrLys Thr Lys Ile Lys Leu Cys Ile Leu 165 170 175 Leu His Ala Phe Arg IleArg Ala Val Thr Ile Asn Arg Met Met Ser 180 185 190 Tyr Leu Asn Ser Ser195 2193 base pairs nucleic acid single linear cDNA 5 AGCACGAGAGCAGAAGAGAC TAGTTTCAGA CCCAGAAAAC TCTGCAGCCT GCCCAGAAGC 60 AAGATGCATCCTCAGCAGCT GGTCATCGCC TGGTTTTACC TGGTTTTGCT GGCACCTCCT 120 CTCATGGCCATATGGGAACT GGAGAAAAAC GTTTATGTTG TAGAGTTGGA CTGGCACCCT 180 GATGCCCCCGGAGAAATGGT GGTCCTCACC TGCAATACTC CTGAAGAAGA TGACATCACC 240 TGGACCTCTGACCAGAGCAG TGAAGTCCTA GGCTCTGGTA AAACTCTGAC CATCCAAGTC 300 AAAGAATTTGCAGATGCTGG CCAGTATACC TGTCATAAAG GAGGCGAGGT TCTGAGCCAT 360 TCGTTCCTCCTGATACACAA AAAGGAAGAT GGAATTTGGT CCACTGATAT CTTAAGGGAA 420 CAGAAAGAATCCAAAAATAA GATCTTTCTA AAATGTGAGG CAAAGAATTA TTCTGGACGT 480 TTCACCTGCTGGTGGCTGAC GGCAATCAGT ACCGATTTGA AATTCACTGT CAAAAGCAGC 540 AGAGGCTCCTCTGACCCCCA AGGGGTGACT TGTGGAGCAG CGACACTCTC AGCAGAGAAG 600 GTCAGAGTGGACAACAGGGA TTATAAGAAG TACACAGTGG AGTGTCAGGA GGGCAGTGCC 660 TGCCCGGCTGCCGAGGAGAG CCTACCCATT GAAGTCGTGG TGGACGCTAT TCACAAGCTC 720 AAGTACGAAAACTACACCAG CAGCTTCTTC ATCAGGGACA TCATCAAACC GGACCCACCC 780 AAGAACCTGCAACTGAAGCC ATTAAAAAAT TCTCGGCATG TGGAAGTGAG CTGGGAATAC 840 CCTGACACCTGGAGCACCCC ACATTCCTAC TTCTCCTTAA CATTTGGCGT ACAGGTCCAG 900 GGCAAGAACAACAGAGAAAA GAAAGACAGA CTCTCCGTGG ACAAGACCTC AGCCAAGGTC 960 GTGTGCCACAAGGATGCCAA GATCCGCGTG CAAGCCAGAG ACCGCTACTA TAGCTCATCC 1020 TGGAGCAACTGGGCATCCGT GTCCTGCAGT TAGGTTCCAA TCCCAGGATG AAACCTTGGA 1080 GGAAAAGTGGAAGATATTAT GCAGAAGTTT TTAAAGAGAC AATGGAATAG GCCCCAAAAG 1140 TTATTTTCTACCTAATTTGC TTTTTGCAAA GGATCATTAT AATGTTTTTG TAGTAGTTTT 1200 ACATTGAAATGCCAAATGCC CACTGAAGCA ATTAGCTACT TTATTTATAG ATTTTCTAGC 1260 TAGCAGGTTGCCACCGACCT TAATGCTATT TAAATATTTA AGTAATTTAT GTATTTATTA 1320 ATTTATTGTTATTGAACACT TCTGTGTCAA GATGTATTGT ATGTTCATAC TCTCAGGACC 1380 TGATCTGTAAGGAATAGGCC CTATTATGCA AAATGTGAAT TTATGTGTTA TTTATACTGA 1440 CAACTTTTCAAACAAGACTA CAAGTGCATC AGTTTTATGA CAACCAGTGA GAACACAGTA 1500 TTCTGATGCCAGCACCAATA ATATGTTTGT GATGGATGGG AACACAGTTA AACAGAAGCA 1560 CGGAGACATGAATCCATTTG AAAAGGTTCT GGTGACCGAG ATGTTAGCTC CTGTGTCCGT 1620 GAAGATTTCCTTGAGGTGGT GTTGCTAAAG CAATTCAGGA CCACCTGCAC TTCTAAGCAA 1680 GTTCAGTTGTTTTATTTTTG TTGTTGGTGG TGAGTTTTTT TGGGGGGGAG GCAGTTGGAT 1740 GCCTGAATTTAGAAAGGACT AAAAAAATAA CTGAAATTGA AATTCAGCTT CAGCTACCCT 1800 GGCAGTCCCCACCTCCATCT ATCTGTAAGA CATCGGAGAG TGACCCAGAG ACATTGGAAG 1860 TGTCTGGAAAGTAAAAAGGT CTTAGGATCC AAGAGGGAGA ACAAGTATAG TACGGCCAAG 1920 CAAACAAAATTGTCAAAATT GCCAGCTGCT TTTTAATAGC CATGCAAGAC AACACAGAGT 1980 TGCAAAGAAAACAATCAAGA ATTGCTTACT CATCAGCATG AGTGAACCTG ACTGGTGGAT 2040 ATGACCGGACAGTGCCAATC ACTAAGGTGC TACTTTTAAG TAATGAATGT GCTTTCTGTA 2100 AAGTGATTTCATTTGTTTTC TGTTTACTTA TTTCTGACAG TGAACTAATA AAAATATAAT 2160 TCTTCTTTGCAATAATAAAA AAAAAAAAAA AAA 2193 921 base pairs nucleic acid single linearcDNA 6 ATATGGGAAC TGGAGAAAAA CGTTTATGTT GTAGAGTTGG ACTGGCACCC TGATGCCCCC60 GGAGAAATGG TGGTCCTCAC CTGCAATACT CCTGAAGAAG ATGACATCAC CTGGACCTCT 120GACCAGAGCA GTGAAGTCCT AGGCTCTGGT AAAACTCTGA CCATCCAAGT CAAAGAATTT 180GCAGATGCTG GCCAGTATAC CTGTCATAAA GGAGGCGAGG TTCTGAGCCA TTCGTTCCTC 240CTGATACACA AAAAGGAAGA TGGAATTTGG TCCACTGATA TCTTAAGGGA ACAGAAAGAA 300TCCAAAAATA AGATCTTTCT AAAATGTGAG GCAAAGAATT ATTCTGGACG TTTCACCTGC 360TGGTGGCTGA CGGCAATCAG TACCGATTTG AAATTCACTG TCAAAAGCAG CAGAGGCTCC 420TCTGACCCCC AAGGGGTGAC TTGTGGAGCA GCGACACTCT CAGCAGAGAA GGTCAGAGTG 480GACAACAGGG ATTATAAGAA GTACACAGTG GAGTGTCAGG AGGGCAGTGC CTGCCCGGCT 540GCCGAGGAGA GCCTACCCAT TGAAGTCGTG GTGGACGCTA TTCACAAGCT CAAGTACGAA 600AACTACACCA GCAGCTTCTT CATCAGGGAC ATCATCAAAC CGGACCCACC CAAGAACCTG 660CAACTGAAGC CATTAAAAAA TTCTCGGCAT GTGGAAGTGA GCTGGGAATA CCCTGACACC 720TGGAGCACCC CACATTCCTA CTTCTCCTTA ACATTTGGCG TACAGGTCCA GGGCAAGAAC 780AACAGAGAAA AGAAAGACAG ACTCTCCGTG GACAAGACCT CAGCCAAGGT CGTGTGCCAC 840AAGGATGCCA AGATCCGCGT GCAAGCCAGA GACCGCTACT ATAGCTCATC CTGGAGCAAC 900TGGGCATCCG TGTCCTGCAG T 921 921 base pairs nucleic acid single linearcDNA 7 ATATGGGAAC TGGAGAAAAA CGTTTATGTT GTAGAGTTGG ACTGGCACCC TGATGCCCCC60 GGAGAAATGG TGGTCCTCAC CTGCAATACT CCTGAAGAAG ATGACATCAC CTGGACCTCT 120GACCAGAGCA GTGAAGTCCT AGGCTCTGGT AAAACTCTGA CCATCCAAGT CAAAGAATTT 180GCAGATGCTG GCCAGTATAC CTGTCATAAA GGAGGCGAGG TTCTGAGCCA TTCGTTCCTC 240CTGATACACA AAAAGGAAGA TGGAATTTGG TCCACTGATA TCTTAAGGGA ACAGAAAGAA 300TCCAAAAATA AGATCTTTCT AAAATGTGAG GCAAAGAATT ATTCTGGACG TTTCACCTGC 360TGGTGGCTGA CGGCAATCAG TACCGATTTG AAATTCACTG TCAAAAGCAG CAGAGGCTCC 420TCTGACCCCC AAGGGGTGAC TTGTGGAGCA GCGACACTCT CAGCAGAGAA GGTCAGAGTC 480GACAACAGGG ATTATAAGAA GTACACAGTG GAGTGTCAGG AGGGCAGTGC CTGCCCGGCT 540GCCGAGGAGA GCCTACCCAT TGAAGTCGTG GTGGACGCTA TTCACAAGCT CAAGTACGAA 600AACTACACCA GCAGCTTCTT CATCAGGGAC ATCATCAAAC CGGACCCACC CAAGAACCTG 660CAACTGAAGC CATTAAAAAA TTCTCGGCAT GTGGAAGTGA GCTGGGAATA CCCTGACACC 720TGGAGCACCC CACATTCCTA CTTCTCCTTA ACATTTGGCG TACAGGTCCA GGGCAAGAAC 780AACAGAGAAA AGAAAGACAG ACTCTCCGTG GACAAGACCT CAGCCAAGGT CGTGTGCCAC 840AAGGATGCCA AGATCCGCGT GCAAGCCAGA GACCGCTACT ATAGCTCATC CTGGAGCAAC 900TGGGCATCCG TGTCCTGCAG T 921 1441 base pairs nucleic acid single linearcDNA 8 GGCACGAGGG AAAGTCCTGC CGCGCCTCGG GACAATTATA AAAATGTGAT CCCCTGGGTC60 GGCTTCCCAC CATCGCCCTC ACCTGCTGCG TCCACCGTCC GGATCCAGCT CCAGCCCAGT 120GTCCGCCCAG TGCCCGCTCA GCATGTGCCC GCCGCGTGGC CTCCTCCTTG TAACCATCCT 180GGTCCTGTTA AACCACCTGG ACCACCTCAG TTTGGCCAGG AACCTCCCCA CACCCACACC 240AAGCCCAGGA ATGTTCCAGT GCCTCAACCA CTCCCAAACC CTGCTGCGAG CCATCAGCAA 300CACGCTTCAG AAGGCCAGAC AAACTCTAGA ATTTTACTCC TGCACTTCCG AAGAGATTGA 360TCATGAAGAT ATCACAAAAG ATAAAACCAG CACAGTGGAG GCCTGCTTAC CACTGGAATT 420AACCATGAAT GAGAGTTGCC TGGCTTCCAG AGAGATCTCT CTGATAACTA ATGGGAGTTG 480CCTGGCCTCC AGAAAGACCT CTTTTATGAC GACCCTGTGC CTTAGCAGTA TCTATGAGGA 540CTTGAAGATG TACCAGGTGG AGTTCAAGGC CATGAATGCA AAGCTGTTAA TGGATCCTAA 600AAGGCAGATC TTTCTGGATC AAAACATGCT GACAGCTATT GATGAGCTGT TACAGGCCCT 660GAATGTCAAC AGTGTGACTG TGCCACAGAA CTCCTCCCTG GAAGAACCAG ATTTTTATAA 720AACTAAAATC AAGCTCTGCA TACTTCTTCA TGCTTTCAGA ATTCGTGCAG TGACCATCAA 780TAGAATGATG AGCTACCTGA ATTCTTCCTA AAAAGCTGAA GTCTCTCCCA ACCTTAAAGC 840CACTTTTACA GAAATGTGAA CCAAAAAAAC AAACAAAAAC AAAACATATA TATATATATA 900TATGTGTGTA TATATATATA TATATATATA TATATATATA TATATATATA TTTCATAGGA 960TGTGGGTTAA GAACCAGGGA GTGGGTGGCT TGACCTGGTC CTCCTTAAGC TAGTACAATA 1020ATTCTCATGC TTGTTTACAT TAGTTGCCAC CCAAAATTTG AAAGATATGA CTGTTATGTC 1080CACATGATGC CTCTGACCAA GTCTATTTCA CATTTACTAG GGAGGGATAA GTTCTTTTTA 1140AGTTTTCATG AGCAAATTGC TAAAGAGGGA AAATGTCCTT CCTTGAACAT GTTTTTCTTT 1200TCCCTTTAAT AGAAAAGCAA GAATTTATAA GCTATTTCTG TACCTAAGTG TTTGTAGACA 1260CAAACACCCC AGCATAATTT ATTTTAAAAT ACTTATTTAT ATAATTTTGT GTTCATGAAA 1320GCATGTGAGC TAACTTATAT TTATTTATGT TATATTTATT AAAATATTTA TTATCCAATG 1380GATTTGAGAA CTACCTTATT TTCTAAAAAT AAAATGATTG AATAAAAAAA AAAAAAAAAA 1440 A1441 591 base pairs nucleic acid single linear cDNA 9 AGGAACCTCCCCACACCCAC ACCAAGCCCA GGAATGTTCC AGTGCCTCAA CCACTCCCAA 60 ACCCTGCTGCGAGCCATCAG CAACACGCTT CAGAAGGCCA GACAAACTCT AGAATTTTAC 120 TCCTGCACTTCCGAAGAGAT TGATCATGAA GATATCACAA AAGATAAAAC CAGCACAGTG 180 GAGGCCTGCTTACCACTGGA ATTAACCATG AATGAGAGTT GCCTGGCTTC CAGAGAGATC 240 TCTCTGATAACTAATGGGAG TTGCCTGGCC TCCAGAAAGA CCTCTTTTAT GACGACCCTG 300 TGCCTTAGCAGTATCTATGA GGACTTGAAG ATGTACCAGG TGGAGTTCAA GGCCATGAAT 360 GCAAAGCTGTTAATGGATCC TAAAAGGCAG ATCTTTCTGG ATCAAAACAT GCTGACAGCT 420 ATTGATGAGCTGTTACAGGC CCTGAATGTC AACAGTGTGA CTGTGCCACA GAACTCCTCC 480 CTGGAAGAACCAGATTTTTA TAAAACTAAA ATCAAGCTCT GCATACTTCT TCATGCTTTC 540 AGAATTCGTGCAGTGACCAT CAATAGAATG ATGAGCTACC TGAATTCTTC C 591 19 amino acids aminoacid single linear peptide 10 Ala Phe Arg Ile Arg Ala Val Thr Ile AsnArg Met Met Ser Tyr Leu 1 5 10 15 Asn Ser Ser 20 amino acids amino acidsingle linear peptide 11 Ile Trp Glu Leu Glu Lys Asn Val Tyr Val Val GluLeu Asp Trp His 1 5 10 15 Pro Asp Ala Pro 20 38 base pairs nucleic acidsingle linear cDNA 12 ATCTGGGARC TSGARAARAA CGTSTACGTS GTSGARCT 38 32base pairs nucleic acid single linear cDNA 13 GGGGTACCGT CGACTCTGACCTTCTCTGCT GA 32 44 base pairs nucleic acid single linear cDNA 14GCTCTAGAAA GCTTGAATTC GTCGACAACA GGGATTATAA GAAG 44 21 base pairsnucleic acid single linear cDNA 15 ATGTGCCCNC CNCTNTGYCT N 21 21 basepairs nucleic acid single linear cDNA 16 ATGTGCCCNC CNTTRTGYTT R 21 21base pairs nucleic acid single linear cDNA 17 RTGNAGNAGD ATGCANAGCT T 2121 base pairs nucleic acid single linear cDNA 18 RTGYAAYAAD ATGCAYAACT T21 10 amino acids amino acid single linear peptide 19 Arg Asn Leu ProThr Pro Thr Pro Ser Pro 1 5 10 21 amino acids amino acid single linearpeptide 20 Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys ThrSer 1 5 10 15 Thr Val Glu Ala Cys 20 16 amino acids amino acid singlelinear peptide 21 Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys IleLys Leu Cys 1 5 10 15 95 base pairs nucleic acid single linear cDNA 22CCGAGCTCGG GTCGACGGCC GCCGCCATGT GCCCCCCCCG TGGCCTCCTC CTGGTGACCA 60TCCTGGTCCT GCTGAACCAC CTGGACCACC TCAGT 95 30 base pairs nucleic acidsingle linear cDNA 23 ACTAGTCTCG AGTTTTTTTT TTTTTTTTTT 30 79 base pairsnucleic acid single linear cDNA 24 TGAAGATATC ACAAAAGATA AAACCAGCACAGTGGAGGCC TGCCTGCCAC TGGAACTGAC 60 CATGAATGAG AGTTGCCTG 79 36 basepairs nucleic acid single linear cDNA 25 AGGATCCATC AGCAGCTTTGCATTCATGGC CTTGAA 36 689 base pairs nucleic acid single linear cDNA 26GCCGCCATGT GCCCCCCCCG TGGCCTCCTC CTGGTGACCA TCCTGGTCCT GCTGAACCAC 60CTGGACCACC TCAGTTTGGC CAGGAACCTC CCCACACCCA CACCAAGCCC AGGAATGTTC 120CAGTGCCTCA ACCACTCCCA AACCCTGCTG CGAGCCATCA GCAACACGCT TCAGAAGGCC 180AGACAAACTC TAGAATTTTA CTCCTGCACT TCCGAAGAGA TTGATCATGA AGATATCACA 240AAAGATAAAA CCAGCACAGT GGAGGCCTGC CTGCCACTGG AACTGACCAT GAATGAGAGT 300TGCCTGGCTT CCAGAGAGAT CTCTCTGATA ACTAATGGGA GTTGCCTGGC CTCCAGAAAG 360ACCTCTTTTA TGACGACCCT GTGCCTTAGC AGTATCTATG AGGACTTGAA GATGTACCAG 420GTGGAGTTCA AGGCCATGAA TGCAAAGCTG CTGATGGATC CTAAAAGGCA GATCTTTCTG 480GATCAAAACA TGCTGACAGC TATTGATGAG CTGTTACAGG CCCTGAATGT CAACAGTGTG 540ACTGTGCCAC AGAACTCCTC CCTGGAAGAA CCAGATTTTT ATAAAACTAA AATCAAGCTC 600TGCATACTTC TTCATGCTTT CAGAATTCGT GCAGTGACCA TCAATAGAAT GATGAGCTAC 660CTGAATTCTT CCTAAAAAGC TGAAGTCTC 689

What is claimed is:
 1. A protein, comprising: (a) an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 2; and (b)an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:4.
 2. The protein of claim 1, wherein either or both of the polypeptidesof said protein are a recombinant polypeptide prepared by using thegenetic recombination technique.
 3. The protein of claim 2, wherein saidprotein has the activity to activate feline lymphocytes.
 4. The proteinof claim 3, wherein said feline lymphocytes are feline cytotoxic Tlymphocytes.
 5. The protein of claim 1, wherein said protein has theactivity to activate feline lymphocytes.
 6. The protein of claim 5,wherein said feline lymphocytes are feline cytotoxic T lymphocytes. 7.An agent for treating feline viral diseases, comprising the protein ofclaim 1 as an active ingredient.
 8. A protein, comprising: (a) anisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2;and (b) an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 4 with deletion, insertion or substitution at any one toseveral amino acid residues, wherein the C terminal of said polypeptidehas the amino acid sequence of SEQ ID NO:
 10. 9. The protein of claim 8,wherein said protein has the activity to activate feline lymphocytes.10. The protein of claim 9 wherein said feline lymphocytes are felinecytotoxic T lymphocytes.
 11. The protein of claim 8, wherein either orboth of the polypeptides of said protein are a recombinant polypeptideprepared by using the genetic recombination technique.
 12. The proteinof claim 11, wherein said protein has the activity to activate felinelymphocytes.
 13. The protein of claim 12, wherein said felinelymphocytes are feline cytotoxic T lymphocytes.
 14. An agent fortreating feline viral diseases, comprising the protein of claim 8 as anactive ingredient.
 15. A protein, comprising: (a) an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 2 withdeletion, insertion or substitution at any one to several amino acidresidues; and (b) an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:
 4. 16. The protein of claim 15, wherein either orboth of the polypeptides of said protein are a recombinant polypeptideprepared by using the genetic recombination technique.
 17. The proteinof claim 16, wherein as the activity to activate feline lymphocytes. 18.The protein of claim 17, wherein said feline lymphocytes are felinecytotoxic T lymphocytes.
 19. The protein of claim 15, wherein as theactivity to activate feline lymphocytes.
 20. The protein of claim 19,wherein said feline lymphocytes are feline cytotoxic T lymphocytes. 21.An agent for treating feline viral diseases, comprising the protein ofclaim 15 as an active ingredient.
 22. A protein, comprising: (a) anisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2with deletion, insertion or substitution at any one to several aminoacid residues; and (b) an isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 4 with deletion, insertion or substitution at anyone to several amino acid residues, wherein the C terminal of saidpolypeptide has the amino acid sequence of SEQ ID NO:
 10. 23. Theprotein of claim 22, wherein either or both of the polypeptides of saidprotein are a recombinant polypeptide prepared by using the geneticrecombination technique.
 24. The protein of claim 23, wherein theprotein has the activity to activate feline lymphocytes.
 25. The proteinof claim 24, wherein said feline lymphocytes are feline cytotoxic Tlymphocytes.
 26. The protein of claim 22, wherein the protein has theactivity to activate feline lymphocytes.
 27. The protein of claim 26,wherein said feline lymphocytes are feline cytotoxic T lymphocytes. 28.An agent for treating feline viral diseases, comprising the protein ofclaim 22 as an active ingredient.